Continued cytoadherence of Plasmodium falciparum infected red blood cells after antimalarial treatment

Abstract

Development of severe disease in Plasmodium falciparum malaria infection is thought to be, at least in part, due to the sequestration of trophozoite-stage infected red blood cells in the microvasculature. The process of cytoadherence is mediated by binding of the parasite protein PfEMP-1 on the surface of infected red blood cells to endothelial cell receptors. Although antimalarial treatments rapidly kill parasites, significant mortality is still seen in severe malaria, particularly within 24h of hospital admission. We find that cytoadherence of infected red blood cells continues for several hours after killing of the parasite by antimalarials; after 24h treatment using a range of antimalarials binding is approximately one-third the level of untreated parasite cultures. This is consistent with the maintained presence of PfEMP-1 on the surface of drug-treated infected red blood cells. A specific advantage of artesunate over other treatments tested is seen on addition of this drug to younger ring stage parasites, which do not mature to the cytoadherent trophozoite-stage. These findings show that cytoadherence, a potential pathogenic property of P. falciparum infected red blood cells, continues long after the parasite has been killed. These data support the development of adjunctive therapies to reverse the pathophysiological consequences of cytoadherence.

Fig. 1

Phenotype of trophozoite-stage ItG iRBC after artesunate treatment. (A) Geimsa stained smears of ItG infected red blood cells after 0, 4, 8, and 24 h treatment with 500 nM artesunate under culture conditions. (B) Adhesion to ICAM-1 protein (50 μg/ml) under flow conditions (0.05 Pa shear stress) after 0, 4, 8 or 24 h treatment with 500 nM artesunate. Mean of three independent experiments ± standard deviation (SD). *P < 0.05 and **P < 0.01 compared to untreated control (c) trophozoites.

Fig. 2

Adhesion of trophozoite-stage ItG iRBC to endothelial cells after 0, 4, 8 or 24 h artesunate treatment. Binding under static conditions to (A) HUVEC and (B) HDMEC after TNF stimulation (1 ng/ml, 18 h) showing bound iRBC per mm² confluent endothelial cells. Binding under flow conditions to (C) HUVEC and (D) HDMEC after TNF stimulation of endothelial cells showing percent of control (untreated trophozoites) binding. Each represents mean of three independent experiments ± SD. *P < 0.05 and **P < 0.01 compared to untreated controls (c).

Fig. 3

Analysis of PfEMP-1 on the surface of live trophozoite-stage A4 iRBC after immunofluorescence using the primary antibody BC6. (A) Confocal fluorescence images of (i) top plane, and (ii) centre plane of BC6 fluorescence, (iii) ethidium bromide labelling of parasite, and phase contrast images (iv), after 0 (upper) 4 (middle) or 24 h (lower) artesunate treatment. Scale bar (2 μM) on phase contrast image. (B) Quantitation of PfEMP-1 levels by flow cytometry analysis after BC6 primary antibody and Alexa488 coupled secondary antibody (FL-1, X-axis) and counterstaining infected red blood cells with ethidium bromide (FL-2, Y-axis). In the density plots uninfected red blood cells are the population in the lower left. Box R1 represents BC6 positive infected red blood cells. Plot (i) shows 0 h culture, plot (ii) shows the culture after 24 h artesunate treatment. (C) Histogram showing quantitation of BC6 positive fluorescence intensity (X-axis) of the infected population (gated as ethidium bromide positive) untreated trophozoites (open black) or after artesunate (500 nM) 24 h (solid red). (D) Mean fluorescence intensity (MFI) for PfEMP-1 positive population (gate R1) mean ± SD for three independent experiments, ** indicates a statistically significant difference compared to controls (P < 0.01) as determined by T-test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 4

Adhesion of trophozoite-stage ItG iRBC to ICAM-1 protein under flow conditions after 4 h (A), 8 h (B) and 24 h (C) treatment with artesunate (art) (500 nM), quinine (quin) (25 μM), lumefantrine (lum) (15 μM) or piperaquine (pip) (100 nM). Results are mean of three independent experiments ± SD. *P < 0.05 and **P < 0.01 as determined by T-test compared to untreated control (c) at each time point. The untreated control for the 24 h time point was a stage matched untreated mid-trophozoite culture indicated by a dotted outline as the untreated original culture had reinvaded to non-cytoadherent ring stages. There was no significant difference in the adhesion of any of the untreated controls at the different time points. Treated cultures were dead pyknotic “trophozoite-stage” parasites.

Fig. 5

(A) Geimsa stained smears of ItG iRBC 24 h after the addition of indicated drug treatment to ring stage (young) parasites. (B) Adhesion to ICAM-1 protein under flow conditions 24 h after addition of treatment to ring stage (young) parasites. The untreated ring stage control (representing the starting culture) shown at the 24 h time point was a stage matched untreated culture indicated by a dotted outline as the untreated original culture had matured to the cytoadherent trophozoite-stage. A P-value <0.05 was obtained for the comparison between artemisinin treated parasites and control trophozoites.