Macrophage polarization drives their ability to fuse with HIV-1 infected T lymphocytes. (A) Experimental design for MDM polarization. Human monocytes were differentiated in presence of GM-CSF (M(GM-CSF)) or M-CSF (M(M-CSF)) for 7 d and incubated for additional 2 d with either lipopolysaccharide and interferon-γ (M(LPS+IFNγ)), interleukin-4 (M(IL-4)), or interleukin-10 (M(IL-10)). (B–F) Analysis of HIV-1 infection of polarized MDMs after a 24 h-co-culture with infected Jurkat cells (B–E) or autologous primary CD4⁺ T cells (F) by flow cytometry (B, C, and F) or fluorescence microscopy (D–E). (B) Representative dot plots for HIV-1 p24 signal in MDMs and gating strategy for selection of infected cells. (C and F) Quantification of MDM infection, normalized to the M(M-CSF) condition. C, n = 9 donors, median ± interquartile range and F, n = 5 donors, mean ± SD. (D–E) Quantification of MDM infection index (D, n = 7 donors) and MDM fusion index (E, n = 7 donors). Median ± interquartile range. (G and H) Analysis of long-term infection of polarized MDMs. After 24 h of co-culture, Jurkat cells were washed, and polarized MDMs were maintained in culture for additional 5 d. (G) Representative microscopy images: F-actin (phalloidin, blue), HIV-p24 (yellow) and nuclei (DAPI, gray). Inserts show magnifications of the white squares. Scale bars, 100 and 50 µm. (H) Quantification of HIV-p24 concentration in the supernatant of infected MDMs (n = 6 donors). Median ± interquartile range. Statistical analyses: Multiple comparison test (F) one-way Anova and Tukey, (C) Kruskal–Wallis and Dunn’s, and (D, E, and H) Friedman and Dunn’s. * P ≤ 0.05; ** P ≤ 0.01.