Fig. 4. Gametocyte transmigration dynamics and vascular leakage.

(A) Time series of mCherry_Hsp70-FLuc_ef1α gametocyte crossing the vascular endothelium in an Flk1-GFP (top panel) or UBC-GFP (bottom panel) transgenic mouse. [Time lapse, 5 frames/s; scale bar, 10 μm.] P, parasite; IV, intravascular; EV, extravascular; C1,2, contact site; T1,2, transmigration. In the top panel series, a gametocyte is exiting the BM vasculature (marked as dotted lines), whereas the bottom panel series are showing a gametocyte entering the BM vasculature. (B) Quantification of observed transmigration events across the BM vascular barrier. Upon injection of synchronized parasite stages, only merozoites and gametocytes show significant levels of transmigration events. (C) Sphericity and deformability of P. berghei parasites. Left: Representative in vivo images across stages of mCherry_Hsp70-FLuc_ef1α parasites within UBC-GFP RBCs in static and flow conditions (scale bar, 5 μm). Right: Deformability indices across stages defined as the ratio of maximum cellular diameter before (static) and during (contracted) transmigration (n = 100; bars represent mean, and error bars represent SD. ***P < 0.001). (D and E) Transmigration and vascular leakage. Mice are injected with synchronous mCherry_Hsp70-FLuc_ef1α parasite stages followed by FITC-dextran inoculation, and images are taken at a rate of 5 fps. Representative images of FITC-dextran fluorescence intensity are shown (D). Quantification of FITC-dextran leakage (representative of peripheral blood including iRBCs and uRBCs), demonstrating a unique transient leakage pattern upon injection of mature gametocytes (E). Note that merozoites/rings (M/R) values are normalized by schizont values because of the observed low level of sustained leakage pattern likely caused by co-injected hemozoin. MFI, mean fluorescence intensity. (F) Gametocyte treatment with Cytochalasin D (C.D.) abrogates extravascular accumulation. mCherry_Hsp70-FLuc_ef1α gametocytes are purified ex vivo and incubated with C.D. (or vehicle control) before washout and intravenous injection into naïve mice. Relative abundance and distance from vessels in the extravascular BM compartment are significantly reduced. IVS, intravascular space; EVS, extravascular space. (G) Treatment of infected mice with sildenafil citrate results in gametocyte accumulation in BM and spleen. Mice treated with phenylhydrazine were infected with mCherry_Hsp70-FLuc_ef1α parasites and treated with sulfadiazine–sildenafil citrate for 3 days (or sulfadiazine only, control). Treatment leads to significantly increased accumulation of gametocytes in the BM and spleen of mice compared to control. In all panels, error bars represent SDs of the mean. *** P < 0.001 and **P < 0.01. N.A., not applicable.