Exosomes containing HIV protein Nef reorganize lipid rafts potentiating inflammatory response in bystander cells
Fig 8
Exosomes from HIV-infected cells and plasma of HIV-infected subjects modify rafts and potentiate inflammation.
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).