Figure 2.
Figure 2. HIV-1 infection of macrophages inhibits phagosomal hydrolytic and oxidative activity and induces a delay in the recruitment of late endocytic markers on phagosomes. Primary human macrophages were infected with HIV-1ADA-VSV-G-WT or mock infected for 8 d before incubation with IgG-opsonized beads for various times. (A) Schematic representation of the detection of hydrolytic activity with DQ-BSA beads and oxidative activity with H2DCFDA-OxyBURST beads. Modified substrates emit at 520 nm (sensor) and calibration fluorochrome emits at 647 nm (calibrator). (B) At different times of incubation, cells were placed on ice, resuspended with cold PBS, fixed, and analyzed by flow cytometry. The number of cells containing beads with a modified sensor fluorescence (R2) was divided by the total number of cells containing beads (positive for calibrator, y axis, R1 + R2). (C and D) Results are expressed as a percentage of cells with phagosomes containing detected oxidative (C) or hydrolytic (D) activity. One representative experiment of at least three is shown (see also Fig. S1). (E–G) Primary human macrophages were infected with HIV-1ADA WT or mock infected for 8 d. The cells were incubated for different times with IgG-SRBCs at 37°C. Macrophages were fixed, permeabilized, and labeled with AMCA-labeled anti–rabbit IgG to detect the total SRBCs (unpublished data), anti-p24 followed by Cy2-labeled anti–goat IgG (top line), anti-LAMP1 (third line) followed by Cy3-labeled anti–mouse IgG, and anti-tubulin followed by Cy5-labeled anti–human IgG (not depicted). Particles internalized in phagosomes are also detectable by phase contrast (second row, red arrows). Merged images (bottom line) show SRBCs in blue, LAMP1 in green, and microtubules in red. Z-stacks of wide-field fluorescent images were acquired, deconvoluted, and treated with ImageJ. Bar, 10 µm; magnification in insets is 2.1× (G). The number of phagosomes positive or negative for LAMP1 was counted for at least 10 cells per condition (E and F). Results are expressed as a percentage of total internal phagosome number ± SEM (>200 phagosomes per condition, repeated in n = 3 independent experiments on different donors). *, P < 0.05.

HIV-1 infection of macrophages inhibits phagosomal hydrolytic and oxidative activity and induces a delay in the recruitment of late endocytic markers on phagosomes. Primary human macrophages were infected with HIV-1ADA-VSV-G-WT or mock infected for 8 d before incubation with IgG-opsonized beads for various times. (A) Schematic representation of the detection of hydrolytic activity with DQ-BSA beads and oxidative activity with H2DCFDA-OxyBURST beads. Modified substrates emit at 520 nm (sensor) and calibration fluorochrome emits at 647 nm (calibrator). (B) At different times of incubation, cells were placed on ice, resuspended with cold PBS, fixed, and analyzed by flow cytometry. The number of cells containing beads with a modified sensor fluorescence (R2) was divided by the total number of cells containing beads (positive for calibrator, y axis, R1 + R2). (C and D) Results are expressed as a percentage of cells with phagosomes containing detected oxidative (C) or hydrolytic (D) activity. One representative experiment of at least three is shown (see also Fig. S1). (E–G) Primary human macrophages were infected with HIV-1ADA WT or mock infected for 8 d. The cells were incubated for different times with IgG-SRBCs at 37°C. Macrophages were fixed, permeabilized, and labeled with AMCA-labeled anti–rabbit IgG to detect the total SRBCs (unpublished data), anti-p24 followed by Cy2-labeled anti–goat IgG (top line), anti-LAMP1 (third line) followed by Cy3-labeled anti–mouse IgG, and anti-tubulin followed by Cy5-labeled anti–human IgG (not depicted). Particles internalized in phagosomes are also detectable by phase contrast (second row, red arrows). Merged images (bottom line) show SRBCs in blue, LAMP1 in green, and microtubules in red. Z-stacks of wide-field fluorescent images were acquired, deconvoluted, and treated with ImageJ. Bar, 10 µm; magnification in insets is 2.1× (G). The number of phagosomes positive or negative for LAMP1 was counted for at least 10 cells per condition (E and F). Results are expressed as a percentage of total internal phagosome number ± SEM (>200 phagosomes per condition, repeated in n = 3 independent experiments on different donors). *, P < 0.05.

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