FIG 5.

Human CD300a interacts with PtdEth and PtdSer associated with DENV particles. (A) 3-sn-PtdCho, 3-sn-PtdEth, or 3-sn-PtdSer was coated onro 96-well plates in the presence of ethanol (vehicle). Wells were incubated with human IgG1-Fc, NK-G2D-Fc, TIM3-Fc, DC-SIGN-Fc, and human or mouse CD300a-Fc (2 μg/ml). The data shown are means ± the SD of two independent experiments performed in duplicate. (B) 293T cells stably expressing TIM1 or human or mouse CD300a were incubated for 20 min at 4°C with pHrodo-labeled mouse thymocytes (1:10 ratio). Efferocytosis was initiated by shifting the cells to 37°C, and the percentages of cells that engulfed apoptotic thymocytes were detected by flow cytometry after 2 h of incubation. Cells kept at 4°C were used as a control for background fluorescence. These experiments were repeated three times in duplicate, and the data shown are means ± the SD from one representative experiment. Significance was calculated by comparison with the percentage of fluorescent parental cells using a two-sample Student t test (ns, nonsignificant; *, P < 0.05). (C and D) Immobilized DENV2 JAM particles (10⁶ FIU) were preincubated for 1 h with increasing concentrations of the PtdSer-specific ligand annexin V (0.31 to 20 μg/ml) (C) or the PtdEth-specific ligand Duramycin (0.625 to 5 μM) (D) prior to the addition of the human or mouse CD300a-Fc or DC-SIGN-Fc (2 μg/ml). The data shown are means ± the SD from three independent experiments performed in duplicate. Significance was calculated by comparing binding without blocking reagents to the binding with the indicated concentration of blocking reagents using a one-way analysis of variance statistical test (ns, nonsignificant; *, P < 0.05; **, P < 0.001; ***, P < 0.0001). (E) Immobilized DENV2 JAM particles (10⁶ FIU) were preincubated for 1 h with Duramycin (5 μM) prior to addition of the human CD300a-FC or the mouse CD300a-Fc or TIM-3-Fc (2 μg/ml). The data shown are means ± the SD from two independent experiments performed in duplicate. Significance was calculated by comparing binding in presence or absence of the Duramycin using a two-sample Student t test (ns, nonsignificant; ***, P < 0.0001).