Unveiling the mechanisms for decreased glutathione in individuals with HIV infection

Abstract

We examined the causes for decreased glutathione (GSH) in individuals with HIV infection. We observed lower levels of intracellular GSH in macrophages from individuals with HIV compared to healthy subjects. Further, the GSH composition found in macrophages from HIV(+) subjects heavily favors oxidized glutathione (GSSG) which lacks antioxidant activity, over free GSH which is responsible for GSH's antioxidant activity. This decrease correlated with an increase in the growth of Mycobacterium tuberculosis (M. tb) in macrophages from HIV(+) individuals. In addition, we observed increased levels of free radicals, interleukin-1 (IL-1), interleukin-17 (IL-17) and transforming growth factor-β (TGF-β) in plasma samples derived from HIV(+) individuals compared to healthy subjects. We observed decreased expression of the genes coding for enzymes responsible for de novo synthesis of GSH in macrophages derived from HIV(+) subjects using quantitative PCR (qPCR). Our results indicate that overproduction of proinflammatory cytokines in HIV(+) individuals lead to increased production of free radicals. This combined with the decreased expression of GSH synthesis enzymes leads to a depletion of free GSH and may lead in part to the loss of immune function observed in HIV patients.

Figure 1

(a) The first step in de novo GSH biosynthesis is rate limiting. Glutamine and cysteine are linked by the homodimeric enzyme glutamine-cysteine ligase, (b) GSS catalyzes the second step in GSH biosynthesis, linking glycine and γ-glutamyl cysteine to form GSH. (c) Oxidized GSH can be converted to free GSH by GSR, utilizing NADPH as a cofactor.

Figure 2

(a) Assay of TGF-β in plasma from HIV-infected and healthy subjects. Plasma samples separated from blood of healthy volunteers and HIV-infected individuals were used for measurement of TGF-β. Levels of TGF-β in the plasma samples were determined by ELISA using assay kits procured from eBioscience. Results in Figure 2 are averages of data collected from eight healthy subjects and twelve individuals with HIV infection. Results show elevated TGF-β in HIV-infected subjects (*P ≤ 0.05). (b) Assay of IL-1 in plasma from HIV-infected and healthy subjects. Plasma samples separated from blood of healthy volunteers and HIV-infected individuals were used for measurement of IL-1. Levels of IL-1 in the plasma samples were determined by ELISA using assay kits procured from eBioscience. Results in (b) are averages of data collected from eight healthy subjects and twelve individuals with HIV infection. Results show elevated IL-1 in HIV-infected subjects (*P ≤ 0.05). (c) Assay of IL-17 in plasma from HIV-infected and healthy subjects. Plasma samples separated from blood of healthy volunteers and HIV-infected individuals were used for measurement of IL-17. Levels of IL-17 in the plasma samples were determined by ELISA using assay kits procured from eBioscience. Results in (c) are averages of data collected from eight healthy subjects and eight individuals with HIV infection. Results show elevated IL-17 in HIV-infected subjects (*P ≤ 0.05).

Figure 3

(a) Assay of IL-1 in macrophage culture supernatants from HIV-infected and healthy subjects. Supernatants from human monocyte-derived macrophages (from healthy and HIV-infected individuals) were assayed for the levels of IL-1 using assay kit from ebioscience. Data in (a) denote means ± SE from eight healthy individuals and nine individuals with HIV infection (*P ≤ 0.05). (b) Assay of TNF-α in macrophage culture supernatants from HIV-infected and healthy subjects. Supernatants from human monocyte-derived macrophages (from healthy and HIV-infected individuals) were assayed for the levels of TNF-α using assay kit from ebioscience. Data in (b) are means ± SE from eight healthy individuals and nine individuals with HIV infection (*P ≤ 0.05). (c) Assay of TGF-β in macrophage culture supernatants from HIV-infected and healthy subjects. Supernatants from human monocyte-derived macrophages (from healthy and HIV-infected individuals) were assayed for the levels of TGF-β using assay kit from ebioscience. Data in (c) represent means ± SE from eight healthy individuals and nine individuals with HIV infection (*P ≤ 0.05). (d) Assay of MDA in macrophage lysates from HIV-infected and healthy subjects. Free radical levels in macrophage lysates from healthy subjects and individuals with HIV infection were determined by measuring the levels of MDA using a colorimetric assay kit from Cayman. Results in (d) are averages of data collected from five healthy subjects and five individuals with HIV infection. These elevated MDA concentrations are indicative of elevated free radical concentrations. High standard error values observed in HIV-infected subjects (a–d) are likely due to the varying stages of HIV infection and antiretroviral treatment in the HIV-infected population.

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 (3 × 10⁵) 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 (*P ≤ 0.05). (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 (*P ≤ 0.05). Free GSH concentrations are decreased in HIV-infected macrophages with respect to healthy macrophages (*P ≤ 0.05). (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 n = 4 HIV and n = 5 healthy. Each experiment was performed in triplicate. Macrophages from HIV-infected subjects demonstrate a markedly decreased ability to control intracellular M. tb  growth.

Figure 5

Relative gene expression for the specified target genes in comparison to the endogenous control ACTB. A comparison of gene expression in macrophages from healthy and HIV-infected subjects demonstrates reduced gene expression for all genes involved in the de novo synthesis of GSH in macrophages from HIV-infected subjects. A greater than 3-fold increase in GSR expression is observed in HIV-infected macrophages. Results are from n = 3 individuals for both healthy and HIV-infected macrophages.

Figure 6

Model illustrating the reasons for decreased levels of GSH in macrophages from individuals with HIV infection.