Phagocytosis and activation of bone marrow-derived macrophages by Plasmodium falciparum gametocytes

Abstract

Background: The innate immune response against various life cycle stages of the malaria parasite plays an important role in protection against the disease and regulation of its severity. Phagocytosis of asexual erythrocytic stages is well documented, but little and contrasting results are available about phagocytic clearance of sexual stages, the gametocytes, which are responsible for the transmission of the parasites from humans to mosquitoes. Similarly, activation of host macrophages by gametocytes has not yet been carefully addressed.

Methods: Phagocytosis of early or late Plasmodium falciparum gametocytes was evaluated through methanol fixed cytospin preparations of immortalized mouse C57Bl/6 bone marrow-derived macrophages treated for 2 h with P. falciparum and stained with Giemsa, and it was confirmed through a standardized bioluminescent method using the transgenic P. falciparum 3D7elo1-pfs16-CBG99 strain. Activation was evaluated by measuring nitric oxide or cytokine levels in the supernatants of immortalized mouse C57Bl/6 bone marrow-derived macrophages treated with early or late gametocytes.

Results: The results showed that murine bone marrow-derived macrophages can phagocytose both early and late gametocytes, but only the latter were able to induce the production of inflammatory mediators, specifically nitric oxide and the cytokines tumour necrosis factor and macrophage inflammatory protein 2.

Conclusions: These results support the hypothesis that developing gametocytes interact in different ways with innate immune cells of the host. Moreover, the present study proposes that early and late gametocytes act differently as targets for innate immune responses.

Keywords: Immortalized mouse C57Bl/6 bone marrow-derived macrophages; Macrophage inflammatory protein 2; Malaria; Nitric oxide; Phagocytosis; Plasmodium falciparum gametocytes; Tumour necrosis factor-alpha.

Fig. 1

Set up of a luminescent assay to evaluate the phagocytosis of late GCT by immortalized mouse C57Bl/6 bone marrow-derived macrophages. a After 2 h incubation with GCT, cells were washed with water (water lysis, striped column) or PBS (no lysis, black column); b BMDM were pre-incubated in the presence (+ CytoD, grey column) or absence (-CytoD, white column) of Cytochalasin D (CytoD) 2 µM for 1 h before incubation with GCT. After 2 h incubation with GCT, the water lysis step was performed and luminescence was measured and expressed as Absolute Luminescent Units (ALU). c BMDM were incubated for different time points with GCT. After the water lysis step luminescence was measured and expressed as ALU. Data are the mean ± sd of 3 independent experiments in quadruplicate. Two-Way ANOVA, Tukey’s multiple comparisons test ****P < 0.0001

Fig. 2

Microscopic observation of phagocytosis of late GCT by immortalized mouse C57Bl/6 bone marrow-derived macrophages. a BMDM were incubated with GCT for 2 h, detached from the plate, spotted on smears by cytospin centrifugation, fixed in absolute methanol, and stained with Giemsa. The arrows indicate GCT inside macrophages. Pictures, from one representative experiment out of three, were taken at ×1000 magnification. b GCT were incubated with 3 µM CellTracker™RedCMTPX Dye for 30 min to stain cytoplasm (red in the picture). BMDM were incubated for 2 h with GCT and fixed with formalin 4 %. BMDM membranes were stained with 10 µg/ml WGA-FITC (Wheat germ agglutinin, DBA Italia) (green in the picture). BMDM nuclei were stained by the DAPI present in the mounting medium (Fluoroshield) (blue in the picture). c Z-stack images were taken on an inverted microscope equipped with a confocal spectral imaging system (Olympus Fluoview 1000, Tokyo, Japan) 600× magnification. Scale bar, 10 µm

Fig. 3

Immortalized mouse C57Bl/6 bone marrow-derived macrophages were treated with early or late GCT at the cell:GCT ratio of 1:1.5, for 2 h. a After washing and lysis, luminescence was read using a Biotek Synergy4 microplate reader. Results are expressed as arbitrary luminescence units (ALU). *p < 0.05; ****P < 0.0001 versus control, One-Way ANOVA, Dunnett’s multiple comparison test. b Linear correlation between the number of GCT and the relative luminescence in two different dose-response curves, one for late GCT, one for early GCT. c Magnification of the lower part of b. The table shows the luminescence emitted (ALU) by the medium alone, or by early or late GCT (BMDM treatment), and the numbers of GCT phagocytized by BMDM extrapolated from the dose-response curve shown in c (GCT number). d BMDM were pre-incubated in the presence (+ CytoD, grey column) or absence (−CytoD, white column) of Cytochalasin D (CytoD) 2 µM for 1 h before incubation with GCT. After 2 h incubation with GCT, the water lysis step was performed and luminescence was measured and expressed as Absolute Luminescent Units (ALU). ***p < 0.001; ****P < 0.0001 versus control, Two-Way ANOVA, Tuckey’s multiple comparison test. Data are the mean ± sd of 3 independent experiments in triplicate

Fig. 4

Immortalized mouse C57Bl/6 bone marrow-derived macrophages were treated with early or late GCT at the cell:GCT ratio of 1:2, for 24 h. The production of TNF and MIP-2 was evaluated in the supernatants by ELISA. Nitric oxide production was evaluated by measuring nitrite in cell supernatants by Griess assay. LPS (100 ng/ml) was used as a positive control. Data are the mean ± SD of 3 independent experiments in triplicate. *p < 0.05; **p < 0.01; ***p < 0.001; ****P < 0.0001 versus control, Two-Way ANOVA, Dunnett’s multiple comparison test

Fig. 5

Immortalized mouse C57Bl/6 bone marrow-derived macrophages were pre-incubated in the presence (+ CytoD, grey column) or absence (-CytoD, white column) of Cytochalasin D (CytoD) 2 µM for 1 h before incubation with early or late GCT at the cell:GCT ratio of 1:2, for 24 h. The production of NO was evaluated by measuring nitrite in cell supernatants by Griess assay. The production of TNF and MIP-2 was evaluated in the supernatants by ELISA. Data are the mean ± sd of 3 independent experiments in triplicate. *p < 0.05; **p < 0.01; ***p < 0.001; ****P < 0.0001 versus control, Two-Way ANOVA, Tukey’s multiple comparison test