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. 2019 Jul 25;15(7):e1007907. doi: 10.1371/journal.ppat.1007907

Fig 8. Exosomes from HIV-infected cells and plasma of HIV-infected subjects modify rafts and potentiate inflammation.

Fig 8

A–The effect of exosomes produced by human monocyte derived macrophages (MDM) infected with HIV (exHIV) or ΔNefHIV (exΔNefHIV) on the abundance of rafts in MDM measured with flow cytometry; 48 h incubation; box plot of n = 6 (six separate experiments with MDM from 6 different donors) is shown; *p<0.05 versus both Mock and exΔNefHIV; B–The effect of exosomes produced by human monocyte derived macrophages infected with HIV (exHIV) or ΔNefHIV (exΔNefHIV) on secretion of TNFα by MDM over 48 h; box plot of n = 6 is shown; **p<0.01 versus both Mock and exΔNefHIV; C–The effect of exosomes produced by human monocyte derived macrophages infected with HIV (exHIV) or ΔNefHIV (exΔNefHIV) on the secretion of IL-6 over 48 h incubation. N = 6, *p<0.05 versus both Mock and exΔNefHIV; D–The effect of exNef, exGFP and exosomes isolated from plasma of subjects infected with HIV (ex(WT-HIV)) or infected with Nef-deficient strain of HIV (ex(ΔNef-HIV)) or uninfected (ex(HIV-)) (15 μg/ml of exosomal protein except when 4 μg/ml is indicated, 48h) on the abundance of lipid rafts in RAW264.7 macrophages. Scale bars 10 μm; E–Quantitation of the effect of exosomes (as in A)) on the abundance of lipid rafts. Mean ± SEM are shown; **p<0.01 versus exGFP and ex(HIV-). F–The effect of exNef, exGFP and exosomes isolated from plasma of subjects infected with HIV (ex(WT-HIV)) or infected with Nef-deficient strain of HIV (ex(ΔNef-HIV)) or uninfected (ex(HIV-)) (15 μg/ml of exosomal protein except when 4 μg/ml is indicated, 48h) on the abundance and localization of lipid rafts (red) and TLR4 (green) in RAW264.7 macrophages. Scale bars 10 μm; G–Quantitation of the effect of exosomes (as in B)) on the total abundance of TLR4 in cells. Mean ± SEM are shown; *p<0.05, **p<0.01 versus exGFP and ex(HIV-). H–Quantitation of the effect of exosomes (as in B)) on the abundance of TLR4 in lipid raft regions. Mean ± SEM are shown; **p<0.01 versus exGFP and ex(HIV-/-); J–The effect of exNef, exGFP and exosomes isolated from plasma of subjects infected with HIV (ex(WT-HIV)) or infected with Nef-deficient strain of HIV (ex(ΔNef-HIV)) or uninfected (ex(HIV-)) (15 μg/ml of exosomal protein except when 4 μg/ml is indicated, 48h) and stimulation with 100 ng/ml LPS on secretion of TNFα by RAW264.7 macrophages over 1 h; Mean ± SEM are shown; **p<0.01, ***p<0.001; I–The effect of exNef, exGFP and exosomes isolated from plasma of subjects infected with HIV (ex(WT-HIV)) or infected with Nef-deficient strain of HIV (ex(ΔNef-HIV)) or uninfected (ex(HIV-)) (15 μg/ml of exosomal protein except when 4 μg/ml is indicated, 48h) and stimulation with 100 ng/ml LPS on secretion of IL-6 by RAW264.7 macrophages over 1 h; Mean ± SEM are shown; **p<0.01, ***p<0.001; K—Western blot for the indicated amounts of rNef and ex(WT-HIV) (μg of exosomal protein).