Abstract
BackgroundPhagocytosis of infected and uninfected erythrocytes is an important feature of malaria infections. Flow cytometry is a useful tool for studying phagocytic uptake of malaria-infected erythrocytes in vitro. However, current approaches are limited by the inability to discriminate between infected and uninfected erythrocytes and a failure to stain the early developmental ring stages of infected erythrocytes. The majority of infected erythrocytes in circulation are of the ring stage and these are therefore important targets to study.Methodology/Principal Findings In vitro P. falciparum cultures comprising infected and uninfected erythrocytes were labeled and exposed to cells derived from the human monocytic THP-1 cell line. Phagocytosis was assayed by flow cytometry. Dual labeling of Plasmodium DNA and erythrocyte cytoplasm with dihydroethidium and CellTrace™ Violet respectively allowed, for the first time, the detection and enumeration of phagocytes with ingested erythrocytes from both early ring- and late schizont-stage P, falciparum cultures. The sensitivity of the method was tested using varying conditions including phagocyte type (monocytes versus macrophages), parasite stage (rings versus schizonts), and negative (incubation with cytochalasin D) and positive (incubation with immune sera) effectors of phagocytosis. The current assay clearly demonstrated uptake of infected and uninfected erythrocytes exposed to phagocytes; the extent of which was dependent on the conditions mentioned.ConclusionsWe describe a simple, sensitive and rapid method for quantifying phagocytosis of P. falciparum-infected erythrocytes, by flow cytometry. This approach can be applied for studying parasite-phagocyte interactions under a variety of conditions. The investigation of phagocytosis of P. falciparum-infected erythrocytes can extend from looking solely at late-staged infected erythrocytes to include early-staged ones as well. It does away with the need to purify infected cells, allowing the study of effects on neighboring uninfected cells. This method may also be translated for use with different types of phagocytes.
Highlights
Malaria is one of the most prevalent epidemic diseases in the world, in the subtropical and tropical regions, with 300 to 500 million new infections and approximately 1 to 2 million deaths annually [1]
The parasitemia obtained via flow cytometry analysis at 5 mg DHE per milliliter was 14.42% which corresponded to the Giemsa smear (Figure1A and Figure S1).Comparing DHE with two other commonly used DNA stains (EB and Hoechst 33342), ring-staged parasite cultures were dually stained with either 5 mg/ml DHE and 1 mg/ml Hoechst 33342 or 10 mg/ml ethidium bromide (EB) [16] and 1 mg/ml Hoechst 33342
At 5 mg/ml, approximately 20% of the Hoechst-stained population was co-stained with EB (Figure 2A) whereas more than 90% erythrocytes in the Hoechst-stained population were costained with DHE (Figure 2B)
Summary
Malaria is one of the most prevalent epidemic diseases in the world, in the subtropical and tropical regions, with 300 to 500 million new infections and approximately 1 to 2 million deaths annually [1]. The control of this disease is hindered by spreading resistance of the malaria parasite, Plasmodium species, to common antimalarials such as the quinolines, the antifolates. The host immune response during a malaria infection involves both innate immunity and adaptive immunity. Innate immunity is important in controlling parasitemia in the acute phase of infection and for initiating adaptive immunity. The majority of infected erythrocytes in circulation are of the ring stage and these are important targets to study
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