Murine malaria parasite sequestration: CD36 is the major receptor, but cerebral pathology is unlinked to sequestration

Abstract

Sequestration of malaria-parasite-infected erythrocytes in the microvasculature of organs is thought to be a significant cause of pathology. Cerebral malaria (CM) is a major complication of Plasmodium falciparum infections, and PfEMP1-mediated sequestration of infected red blood cells has been considered to be the major feature leading to CM-related pathology. We report a system for the real-time in vivo imaging of sequestration using transgenic luciferase-expressing parasites of the rodent malaria parasite Plasmodium berghei. These studies revealed that: (i) as expected, lung tissue is a major site, but, unexpectedly, adipose tissue contributes significantly to sequestration, and (ii) the class II scavenger-receptor CD36 to which PfEMP1 can bind is also the major receptor for P. berghei sequestration, indicating a role for alternative parasite ligands, because orthologues of PfEMP1 are absent from rodent malaria parasites, and, importantly, (iii) cerebral complications still develop in the absence of CD36-mediated sequestration, dissociating parasite sequestration from CM-associated pathology. Real-time in vivo imaging of parasitic processes may be used to evaluate the molecular basis of pathology and develop strategies to prevent pathology.

Fig. 1.

Analysis of luciferase expression in blood stages. Luciferase expression (RLU%, percentage of relative luminescence) of the blood stages during synchronized blood-stage development in vitro and in vivo of PbGFP-LUC_CON (Left) and PbGFP-LUC_SCH (Right). (i and ii) Black lines indicate luciferase expression during one cell cycle (24 h) in vitro. In PbGFP-LUC_CON, expression starts in young trophozoites (10 h), peaks in old trophozoites/young schizonts (16-20 h), and decreases in mature schizonts (24 h). In PbGFP-LUC_SCH, expression occurs exclusively in maturing schizonts (20-24 h). Red lines, luciferase expression in the tail blood of mice during the first 30 h after the injection of merozoites. In PbGFP-LUC_CON, the decrease at ≈20 h is the result of the disappearance of the schizonts from the blood circulations (iii). In PbGFP-LUC_SCH, activity is observed only after 24 h, when newly invaded young ring stages are present (iv) that still show luciferase activity from expression in the schizonts. (iii and iv) Course of parasitemia in tail blood during the first 30 h after injecting merozoites. The drop in parasitemia at 18-22 h results from the disappearance of the schizonts from the peripheral circulation, and the increase after 24 h results from the presence of newly invading ring forms.

Fig. 2.

Visualization of the distribution of luciferase-expressing parasites in live rodents and isolated organs. The distribution of parasites was visualized by measuring luciferase activity in live animals and dissected organs by using an I-CCD video camera. Rainbow images show the relative level of luciferase activity ranging from low (blue), to medium (green), to high (yellow, red). Note that the scale of total photon counts and the time of exposure can be different within separate illustrations. In synchronous infections, measurements were performed at different time points (h, hour) after the injection of purified merozoites. In asynchronous infections, measurements were performed between midnight and 4 a.m. on different days (d) after the injection of 10⁵-10⁶ parasites. 1, adipose tissue; 2, spleen; 3, liver; 4, lungs; 5, heart; 6, kidneys; 7, brain; 8, mammary fat pad; 9, muscles; and 10, testis; see also Fig. 7. (A) Synchronous infection of PbGFP-LUC_CON in Swiss mice (60 s). (B) A Swiss mouse 5 h after injection with nonsequestering gametocytes of PbGFP-LUC_GAM (60 s). (C) Synchronous infection of PbGFP-LUC_SCH in Swiss mice [(Upper 10 s) and (Lower 60 s)]. (D) Asynchronous infection of PbGFP-LUC_SCH in a Swiss mouse at day 7 after infection, just before cerebral complications start (30 s for whole body and 10 s for organs). (E) Synchronous infection of PbGFP-LUC_SCH in G. surdaster (30 s for whole body and 60 s for organs).

Fig. 3.

Visualization of sequestration of schizonts in adipose tissue. Visualization of sequestered schizonts in paraformaldehyde-fixed sections (3 μm) of adipose tissue (from belly fat) of Swiss mice (1, 3, and 4) and G. surdaster (2). Representative sections of adipose tissue are shown from animals at 22 h after the injection of purified schizonts (PbGFP-LUC_SCH), when the sequestration of schizonts occurs. Schizonts are visualized by Giemsa staining (1, 3) or by polarization microscopy (2, 4), demonstrating the characteristic pigment clusters of schizonts that show a marked birefringence by polarization microscopy (magnification, ×660). (5) The presence of GFP-expressing sequestered schizonts (arrows) are shown in unfixed adipose tissue of Swiss mouse (magnification, ×400); see also Fig. 8.

Fig. 4.

Visualization and quantification of the sequestration of P. berghei in synchronized infections in CD36-deficient rodents. The distribution of schizonts (PbGFP-LUC_SCH) was visualized by measuring luciferase activity in live animals and dissected organs by using an I-CCD video camera. Measurements were performed at different time points (h, hour) after the injection of purified merozoites when sequestration of schizonts occurs. 1, adipose tissue; 2, spleen; 3, liver; 4, lungs; 5, heart; 6, kidneys; 7, brain; 8, mammary fat pad; 9, muscles, and 10, testis; see also Figs. 9, 10, and 11. (A) Control CD36^+/+ WKY rat (i, 30 s; iii, 60 s; and v, 30 s)and CD36-deficient SHR rat (ii, 60 s; iv, 30 s; and v, 30 s).(B) Control CD36^+/+ (i, 30 s; iii, 30 s; and v, 10 s) and CD36^-/- C57BL/6 mice (ii, 30 s; iv, 30 s; and v, 10 s). (C) Quantification of the luciferase activity in different organs by using the programs living image and igor pro. Photon counts from intact organs (dissected at 22 h) were expressed as the percentage of the total photon counts of all organs (relative luminescence); shaded box, Swiss mice; black box, control CD36^+/+, and open box: CD36^-/- mice. (Inset) The percentage of schizonts in the peripheral blood (tail blood) circulation of the total number of schizonts present in the animals at 22 h. % RLU, percentage of relative luminescence.

Fig. 5.

Cerebral complications and sequestration of P. berghei in asynchronous infections in CD36^+/+ and in CD36^-/- C57BL/6 mice. (A) Cumulative death of groups of mice as a result of cerebral complications (♦, CD36^+/+; □, CD36^-/-) in three experiments. (B) The course of parasitemia. (Inset) The percentage of schizonts in total irbc in the peripheral blood (tail blood) of control CD36^+/+ and CD36^-/- mice infected with 10⁵-10⁶ parasites (▪, CD36^+/+;▵, CD36^-/-). (C) The distribution of schizonts (PbGFP-LUC_SCH) as visualized by measuring luciferase activity in dissected organs by using an I-CCD video camera (IVIS, Xenogen). Measurements were performed at days 4 and 7 after infection (10 s). 1, adipose tissue; 2, spleen; 3, liver; 4, lungs; 5, heart; 6, kidneys; and 7, brain. (D) Quantification of the luciferase activity (%RLU, percentage of relative luminescence) in different organs (dissected at day 4 or day 7 after infection) of control CD36^+/+ (black box) and CD36^-/- (open box) C57BL/6 mice; see also Figs. 10-12.