Glutathione and growth inhibition of Mycobacterium tuberculosis in healthy and HIV infected subjects

A total of 20 subjects (10 healthy volunteer controls and 10 patients with HIV infection) were enrolled at UMDNJ-University Hospital of Newark and the NJ Medical School, in Newark, NJ. Subjects with HIV infection without tuberculosis (n = 10) were recruited at the Infectious Disease Clinic of UMDNJ-University Hospital. The Clinic is the site of several ongoing studies of HIV treatment; these studies provide anti-retroviral treatment (ART) to enrolled subjects without charge. Patient care was not altered by participation in this study. Patients were defined as being HIV-positive on the basis of a positive ELISA with a confirmatory Western Blot performed as part of their routine care in the clinic. The average CD4 numbers for HIV patients in this study was 423 ± 83/cumm. Only one patient had CD4 counts below 200/cumm. Seven patients were on ART and three patients were not on any treatment at the time of blood draw. Healthy subjects without HIV infection or a history of TB were recruited from the hospital and the university faculty and staff (n = 10). Healthy and HIV-positive subjects with a history of a positive tuberculin test (TST) were excluded from the study so as to maintain strict study definitions. This is according to the CDC recommendation that recognizes that a positive TST reflects latent TB infection.

M. tuberculosis H37Rv was grown in 7H9 with albumin-dextrose complex (ADC). Static cultures of mycobacteria at peak logarithmic phase of growth (between 0.5 and 0.8 at A 600) were used for infection. The bacterial suspension was washed and resuspended in RPMI containing AB serum. Bacterial clumps were disaggregated by vortexing five times with 3-mm sterile glass beads. The bacterial suspension was passed through a 5 μm filter to remove any further clumps. The total number of organisms in the suspension was determined by plating. Processed mycobacteria were frozen as stocks at -80°C. At the time of infection, frozen stocks of processed mycobacteria were thawed and used for macrophage infection.

Human monocyte-derived macrophages (HMDM) were used to study the effects of IFN-γ and GSH in inducing intracellular killing of H37Rv. These experiments were performed only in blood samples from healthy subjects due to the non-availability of sufficient blood volume from HIV patients. Forty ml of blood from healthy subjects were used for monocyte isolation. PBMC were isolated by ficoll hypaque density centrifugation. PBMC were washed with PBS and resuspended in RPMI containing 5% AB serum. PBMC (10 × 10⁶/ well) were distributed into Poly-DL-lysine coated 12 well plates and incubated overnight at 37°C, 5% CO₂ in a humidified atmosphere, to allow monocytes to adhere to the plate. Non-adherent cells were removed by gentle washing and the adherent monocytes were cultured in RPMI containing 5% AB serum for 7 days before being used for infection experiments to allow differentiation to macrophages. The total number of macrophages per well (on day seven) was quantitated by detaching the macrophages from a single well by the addition of ice-cold accutase (Sigma). Viable detached macrophages were counted in a Neubauer counting chamber by trypan blue dye exclusion. The average number of macrophages per well on day 7 is approximately 5 × 10⁵.

IFN-γ is considered a predominant activator of microbicidal functions in macrophages and is essential for prevention of uncontrolled progression of M. tuberculosis infection [2, 18, 27]. We therefore studied the survival of H37Rv in IFN-γ, LPS treated HMDM. HMDM were maintained in vitro and infected with H37Rv, as described previously. H37Rv-infected HMDM were treated with IFN-γ (100 U/ml) and LPS (1 μg/ml), the cultures were terminated at 4 h and 7 days after infection and treatment, to determine the intracellular viability of H37Rv inside unstimulated and IFN-γ, LPS-stimulated macrophages.

Mycobacteria added to heparinized blood (after dilution with tissue culture medium), are rapidly ingested by monocytes and other phagocytic cells such as neutrophils. This model differs from other intracellular infection models in that all blood elements are represented. Interactions of infected monocytes with natural killer cells and antigen-specific T cells result in control of intracellular growth. In contrast to the studies with isolated macrophages, the whole blood assay requires a low volume of blood. Blood was diluted at the following proportion: 300 μl of blood from healthy subjects and patients were diluted to 1 ml with RPMI. Blood cultures were infected with 10⁵ CFU of H37Rv. GSH levels in blood cultures were altered using agents such as NAC (10 mM) and BSO (500 μM) that specifically increase and decrease intracellular GSH. The effect of altered GSH levels on M. tuberculosis survival was studied. Treatment of cells with BSO causes inhibition of GSH synthesis. BSO specifically inhibits the activity of γ-glutamyl-cysteinyl synthetase enzyme, that catalyses the first step reaction in the synthesis of GSH. Blood cultures were treated with either NAC or combination of NAC and BSO for 24 h prior to infection. H37Rv infected whole blood cultures were incubated at 37°C and harvested at selected intervals (2 h and, 48 h) by sedimentation at 2000 rpm for 10 min. Supernatants were used to determine cytokine levels and extracellular mycobacterial growth. Host cells were disrupted by addition of sterile water. The lysates were plated on 7H11 medium enriched with ADC for mycobacterial colonies.

Intracellular GSH levels in PBMC, red blood cells (RBC), and plasma, from healthy individuals and HIV positive subjects were assayed by spectrophotometry, using a GSH assay kit procured from Calbiochem. This approach is used to determine whether GSH levels are decreased in all blood components or just in some specific components. Plasma and cell lysates of RBC and PBMC, derived from healthy and HIV positive subjects, were mixed with equal volume of ice cold 5% metaphosphoric acid (MPA) and centrifuged at 3000 rpm for 15 minutes. Supernatants were used for GSH assay, as per the manufacturer's instruction. Plasma, RBC, and PBMC were separated from whole blood by density gradient centrifugation using ficoll hypaque. Samples were also used for protein assay by Bradford's method using Bio Rad reagent.

Blood cultures were prepared by afore mentioned methods. Blood cultures from healthy subjects and HIV patients were treated as follows: no treatment, infection with H37Rv, and infection with H37Rv and treatment with NAC. Cultures were terminated at 2 h and 48 h, after infection. Uninfected cultures were terminated at the same time points. Cultures were centrifuged at 2000 rpm for 10 min. Cell free supernatants from healthy and HIV patients were used for the cytokine assay, which was performed using a Beadlyte kit procured from Upstate. This is a highly sensitive kit that can be used to detect multiple cytokines in tissue culture samples. A monoclonal antibody specific for a cytokine is covalently linked to a fluorescent bead set, which captures the cytokine. A complementary biotinylated monoclonal cytokine antibody then completes the immunological sandwich and the reaction is detected with streptavidin-phycoerythrin using a Luminex. The assay was performed as per the manufacturer's protocol.

Statistical analysis of the data was carried out using Statview program and the statistical significance was determined using unpaired t test. Data from cytokine assays was analyzed by non-parametric test (Kruskal-wallis). Differences were considered significant at a level of p < 0.05.

We studied the survival of H37Rv in HMDM from healthy subjects. H37Rv-infected HMDM were treated with IFN-γ (100 U/ml) and LPS (1 μg/ml), and the intracellular viability of H37Rv inside unstimulated and IFN-γ, LPS-stimulated macrophages was compared. Figure 1a shows results from six different subjects performed in triplicate. We observed significant growth of H37Rv inside unstimulated HMDM between 1 h and 7 days (Fig 1a). The increase was almost four fold. Stimulation of HMDM cells with IFN-γ, LPS also resulted in significant growth of intracellular H37Rv (Fig 1a). However, the increase in H37Rv growth was less than three-fold (Fig 1a). To examine whether GSH plays a major role in human macrophage killing of H37Rv, HMDM from healthy volunteers were treated with 5, 10, 15, and 20 mM NAC, and intracellular growth of H37Rv was measured. Experiments performed in six different subjects show that treatment of HMDM with 10 mM NAC resulted in stasis in H37Rv growth in three out of six subjects (Fig 1b). Treatment of HMDM with NAC at 5 mM and 15 mM induced growth inhibition of H37Rv, in one out of six, and two out of six subjects, respectively (data not shown). Treatment with 20 mM NAC had no effect on growth inhibition of H37Rv (data not shown). Therefore, NAC at 10 mM is more effective in inducing growth control of M. tuberculosis as compared to IFN-γ, LPS, or other concentrations of NAC (Fig 1b) in isolated HMDM.

Several studies indicate that direct cell contact is required for induction of antimycobacterial activity in human macrophages [6, 33], and that this activity reflects caspase-mediated induction of apoptosis, triggering of toll-like receptors, the release of antibiotic peptides (e.g., granulysin), or unknown mechanisms [4, 36]. Mycobacteria are rapidly ingested by phagocytic cells when added to heparinized blood (after dilution with tissue culture medium). This model differs from other intracellular infection models in that all blood elements are represented.

We therefore tested whether interaction of monocytes with other immune cells will lead to growth inhibition of intracellular H37Rv using whole blood cultures, which provides a micro-environment that is conducive for cellular interactions.

Blood from healthy volunteers was diluted as described and treated with none or 10 mM NAC. The blood cultures were then infected with 5 × 10⁵ CFU of processed H37Rv. Infected blood cultures were terminated at 2 h and 48 h after infection to determine the intracellular viability of H37Rv. Cell suspensions were centrifuged to separate the cell free supernatants and pellets. Supernatants were diluted and plated for extracellular bacterial growth. Intracellular viability of H37Rv was determined by plating the diluted blood cell lysates on 7H11. Infection of blood cultures with H37Rv resulted in almost two-fold increases in the intracellular growth of H37Rv (Fig 1c). The increase in H37Rv growth was statistically significant. Treatment of blood cultures with NAC (10 mM), caused growth inhibition of H37Rv in all seven individuals tested (Fig 1c). The data in Fig 1c are averages from seven healthy subjects. Treatment of cultures with BSO abrogated the growth inhibition effect of NAC (Fig 1c). These results indicate that growth inhibition of H37Rv in NAC treated blood cultures is due to combination of direct antimycobacterial effects of GSH and activation of immune cells induced by GSH.

Intracellular GSH levels in PBMC and RBC were assayed by spectrophotometry as described. We observed a significant and more than 50% decrease in intracellular GSH levels in PBMC (Fig 2a) and RBC (Fig 2b) from HIV patients compared to healthy subjects. Data shown in Fig 2 are averages from six healthy and six HIV-infected subjects. We observed no difference in the plasma GSH levels between healthy and HIV patients.

Intracellular growth of H37Rv was monitored in blood cultures of HIV-positive subjects. We observed a significant growth of H37Rv in unstimulated blood cultures (Fig 3a). NAC treatment induced growth inhibition of intracellular of H37Rv. Data in Fig 3b are averages from data obtained from eight different HIV-positive subjects. BSO treatment abrogated the inhibitory effect brought about by NAC treatment (Fig 3c).

Cytokines were measured in blood culture supernatants from healthy and HIV-infected subjects. Interestingly, in HIV subjects, H37Rv infection induced the blood cultures to produce increased levels of pro-inflammatory cytokines such as IL-1, TNF-α, and IL-6 (Fig 4a, 4b, 4c). H37Rv infection induced almost three fold increases in IL-1 production, compared to uninfected controls (Fig 4a). NAC treatment of H37Rv infected cultures down-regulated IL-1 levels (Fig 4a). Compared to uninfected controls, H37Rv infection induced seven fold increases in TNF-α levels in two patients tested (Fig 4b). NAC treatment of H37Rv infected cultures caused reduction in TNF-α levels (Fig 4b). H37Rv infection induced almost ten fold increases in IL-6 production, in three patients tested (Fig 4c). NAC treatment reduced the levels of IL-6 to those found in the uninfected control. We also observed that infection of HIV blood cultures with H37Rv caused six fold increases in IFN-γ production in two patients and three fold increases in one patient (Fig 4d). In comparison to untreated controls, NAC treatment of H37Rv infected cultures induced ten fold increases in IFN-γ production in two patients and almost four fold increases in one patient (Fig 4d). In summary, our studies show that NAC treatment down-regulated the synthesis of IL-1, IL-6, and TNF-α and increased the levels of IFN-γ (Fig 4a, 4b, 4c, 4d).

With the exception of IL-10, all other cytokines produced by healthy subjects showed no clear trend. The regulation of IL-10 synthesis in response to H37Rv infection and NAC treatment was similar in healthy subjects and HIV patients. H37Rv induced almost ten-fold increases in IL-10 levels in both healthy and HIV-infected subjects (Fig 5a, 5b). Furthermore, NAC treatment reduced the levels of IL-10 to those found in uninfected controls, in both healthy subjects and HIV patients (Fig 5a, 5b).