Research Article

Unveiling the Mechanisms for Decreased Glutathione in Individuals with HIV Infection

Figure 4

(a) Assay of total GSH concentrations in macrophage lysates from HIV-infected and healthy subjects. GSH levels were measured in isolated macrophages from healthy subjects and individuals with HIV infection by spectrophotometry using an assay kit from Arbor Assays. Briefly, an equal volume of ice cold 5% 5-SSA was added to the macrophage ( ) pellet. Supernatants collected after centrifugation were analyzed for total, and oxidized GSH as per manufacturer’s instructions. All GSH measurements were normalized with total protein levels. Data in (a) represent means ± SE from five different healthy and HIV-infected individuals. GSH concentrations are decreased in HIV-infected macrophages with respect to healthy macrophages (* ). (b) Assay of free GSH concentrations in macrophage lysates from HIV-infected and healthy subjects. Free GSH was calculated by subtracting measured oxidized GSH concentrations from the measured total GSH concentrations, per the manufacturer’s instructions. Data in (b) denote means ± SE from five different healthy and HIV-infected individuals. GSH concentrations are decreased in HIV-infected macrophages with respect to healthy macrophages (* ). Free GSH concentrations are decreased in HIV-infected macrophages with respect to healthy macrophages (* ). (c) A comparison of the composition of the total GSH in healthy and HIV-infected subjects reveals that the majority of GSH in HIV-infected macrophages exists as oxidized GSH (~70%), whereas healthy macrophages contain a more balanced GSH makeup (~40% oxidized, ~60% free). (d) Intracellular growth of M. tb in macrophages from healthy and HIV-infected individuals at 1 hour, and 5 days postinfection. Human monocyte-derived macrophages (from healthy and HIV-infected individuals) were infected with the processed H37Rv at a multiplicity of infection of 10 : 1. Infected macrophages were terminated at 1 hour and 5 days postinfection to determine the intracellular survival of H37Rv inside macrophages from healthy and HIV-infected individuals. Macrophage lysates were plated on 7H11 medium enriched with ADC to estimate the growth or killing of H37Rv. Results shown in (d) are averages from HIV and healthy. Each experiment was performed in triplicate. Macrophages from HIV-infected subjects demonstrate a markedly decreased ability to control intracellular M. tb  growth.
734125.fig.004a
(a)
734125.fig.004b
(b)
734125.fig.004c
(c)
734125.fig.004d
(d)