Sodium thiosulfate attenuates glial-mediated neuroinflammation in degenerative neurological diseases

The human monocyte THP-1 and astrocytoma U373 cell lines were obtained from the American Type Culture Collection (Manassas, VA). The human neuroblastoma SH-SY5Y cell line was a gift from Dr R. Ross, Fordham University, NY. These cells were grown in DMEM/F12 medium containing 10 % fetal bovine serum (FBS) and 100 IU/mL penicillin and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA) under humidified 5 % CO₂ and 95 % air.

Human astroglial and microglial cells were isolated from surgically resected temporal lobe tissue as described previously [15]. Briefly, tissues were rinsed with a phosphate-buffered saline (PBS) solution and chopped into small (<2 mm³) pieces with a sterile scalpel. They were incubated in 10 mL of a 0.25 % trypsin solution at 37 °C for 20 min. Subsequently, DNase I (from bovine pancreas, Pharmacia Biotech, Baie d’Urfé, PQ, Canada) was added to reach a final concentration of 50 μg/mL. Tissues were incubated for an additional 10 min at 37 °C. After centrifugation at 275g for 10 min, the cell pellet was resuspended in the serum-containing medium and passed through a 100-μm nylon cell strainer (Becton Dickinson, Franklin Lakes, NJ). The cell suspension was then centrifuged (275g for 10 min), resuspended into 10 mL of Dulbecco’s modified Eagle medium (DMEM)-F12 with 10 % FBS containing gentamicin (50 μg/mL), and plated onto tissue culture plates (Becton Dickinson) in a humidified 5 % CO₂, 95 % air atmosphere at 37 °C for 2 h. This achieved adherence of microglial cells. The non-adherent astrocytes along with myelin debris were transferred into new culture plates. Astrocytes adhered slowly and were allowed to grow by replacing the medium once a week. New passages of cells were generated by harvesting confluent astrocyte cultures using a trypsin–EDTA solution (0.25 % trypsin with EDTA, Invitrogen, Carlsbad, CA). Human astrocytes from up to the fifth passage from four surgical cases were used in the study.

For estimating the purity of astrocytic and microglial cell cultures, aliquots of the cultures were placed on glass slides at 37 °C for 48 h. The attached cells were then fixed with 4 % paraformaldehyde for 1 h at 4 °C and permeablized with 0.1 % Triton X-100 for 1 h at room temperature. After washing twice with PBS, the astrocytic culture slides were treated with a monoclonal anti-GFAP antibody (1/4,000, DAKO) and the microglial slides with the polyclonal anti-Iba-1 antibody (1/500, Wako Chemicals, Richmond, VA) for 3 h at room temperature. The slides were then incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Invitrogen, 1:500) and Alexa Fluor 546-conjugated goat anti-rabbit IgG antibody (Invitrogen, 1:500) in the dark for 3 h at room temperature to yield red fluorescence for Iba-1 positive cells and green fluorescence for GFAP positive cells. To visualize all cells, the slides were washed twice with PBS. Images were acquired using an Olympus BX51 microscope and a digital camera (Olympus DP71). Fluorescent images were colocalized with ImagePro software (Improvision Inc., Waltham, MA). We randomly chose 30 microscopic fields. Each field contained a total of about 500 cells. The numbers of astrocytes which appeared in microglial culture fields averaged 3.11 ± 0.12 cells per field. The numbers of microglia appearing in astrocytic culture fields was 2.05 ± 0.34 cells per field. The purity of microglia and astrocytic cultures was more than 99 % (Fig. 1).

To achieve SH-SY5Y differentiation, the undifferentiated cells were treated for 4 days with a high concentration of retinoic acid (RA, 5 μM) in DMEM/F12 medium containing 5 % FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin [16]. The RA-including medium was changed every 2 days. Differentiated SH-SY5Y cells demonstrated neurite extension, indicative of their differentiation [17], and act like a fully-differentiated human neuron-like cells [18].

The viability of SH-SY5Y cells following incubation with glial cell supernatants was evaluated by MTT assays as previously described [19]. Briefly, the viability was determined by adding MTT to the SH-SY5Y cell cultures to reach a final concentration of 1 mg/mL. Following a 1 h incubation at 37 °C, the dark crystals formed were dissolved by adding a SDS/DMF extraction buffer (300 μL, 20 % sodium dodecyl sulfate, 50 % N,N-dimethylformamide, pH 4.7). Subsequently, plates were incubated overnight at 37 °C, and optical densities at 570 nm were measured by transferring 100 μL aliquots to 96-well plates and using a plate reader with a corresponding filter. Data are presented as a percentage of the values obtained from cells incubated in fresh medium only.

The viability of differentiated SH-SY5Y cells was investigated by the lactate dehydrogenase (LDH) release assay [19]. Briefly, cell culture supernatants (100 μl) were pipetted into the wells of 96-well plates, followed by the addition of 15 μl lactate solution (36 mg/ml in PBS) and 15 μl p-iodonitrotetrazolium violet (INT) solution (2 mg/ml in PBS). The enzymatic reaction was started by addition of 15 μl of NAD⁺/diaphorase solution (3 mg/ml NAD⁺; 2.3 mg solid/ml diaphorase). After 1 h, optical densities were measured with a Model 450 microplate reader (Bio-Rad Laboratories, Richmond, CA) using a 490-nm filter. The amount of LDH released was expressed as a percentage of the value obtained in comparative wells where cells were 100 % lysed by 1 % Triton X-100.

Cytokine levels were measured in cell-free supernatants following 48 h incubation of THP-1 cells, U373 cells, microglial cells, and astrocytes. The cell stimulation protocols in these experiments were the same as that used in protocol 1. Quantitation was performed with ELISA detection kits (Peprotech, NJ) following protocols described by the manufacturer.

For determining TNFα and IL-6 depletion, protocols published in earlier studies were followed [17, 20]. Briefly, microglia were exposed to LPS/IFNγ, and astrocytes to IFNγ, for 2 days. Their supernatants were transferred to 24-well plates which had been coated with anti-TNFα or anti-IL-6 antibodies (10 mg/ml). After 3-h incubation, the supernatants were transferred to SH-SY5Y cells. After incubation at 37 °C for 3 days, MTT assays were performed on the SH-SY5Y cells.

Western blotting on cell lysates was performed as described previously [19]. Briefly, microglia and astrocytes were exposed to NaSH and STS at 100 μM for 8 h and were subsequently exposed to stimulants for 2 h. Human microglia and astrocytes were treated with a lysis buffer (150 mm NaCl, 12 mm deoxycholic acid, 0.1 % Nonidet P-40, 0.1 % Triton X-100, and 5 mm Tris-EDTA, pH 7.4). The protein concentration of the cell lysates was then determined using a BCA protein assay reagent kit (Pierce, Rockford, IL). Proteins in each sample were loaded onto gels and separated by 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (150 V, 1.5 h). The loading quantities of lysate proteins were 100 μg. Following SDS-PAGE, proteins were transferred to a PVDF membrane (Bio-Rad, CA) at 30 mA for 2 h. The membranes were blocked with 5 % milk in PBS-T (80 mm Na₂HPO₄, 20 mm NaH₂PO₄, 100 mm NaCl, 0.1 % Tween 20, pH 7.4) for 1 h and incubated overnight at 4 °C with a polyclonal anti-phospho-P38 MAP kinase antibody (9211, Cell Signaling, Beverly, MA, 1/2,000) or anti-phospho-P65 NFκB antibody (3031, Cell Signaling, 1/1,000). The membranes were then treated with a horseradish peroxidase-conjugated anti-IgG (P0448, DAKO, Mississauga, Ontario, CA, 1:2,000) or the secondary antibody anti-mouse IgG (A3682, Sigma, 1/3,000) for 3 h at room temperature, and the bands were visualized with an enhanced chemiluminescence system and exposure to photographic film (Hyperfilm ECL™, Amersham Biosciences). Equalization of protein loading was assessed independently using α-tubulin as the housekeeping protein. The primary antibody was anti-α-tubulin (T6074, Sigma, 1/2,000) and the secondary antibody anti-mouse IgG (A3682, Sigma, 1/3,000). Primary antibody incubation was overnight at 4 °C, and the secondary antibody incubation was for 3 h at room temperature.

H₂S (HS⁻) levels were measured using a previously described method [15]. To suppress endogenous production of H₂S by CBS, all experiments were done with 0.1 mM of the specific CBS inhibitor hydroxylamine added to the solutions. Two sets of experiments were conducted: one in which the THP-1 cells and U373 cells were unstimulated and a second where they were stimulated with inflammatory mediators for 48 h. For THP-1 cells, the stimulation was LPS at 1 μg/ml and IFNγ at 333 U/ml, and for U373 cells, it was IFNγ at 150 U/ml. The cells in each case were treated with hydroxylamine plus NaSH or hydroxylamine plus STS (100 μM each) for 2, 4, 8, and 12 h. Following treatment, they were homogenized in 250 μl of ice-cold 100 mM potassium phosphate buffer (pH 7.4) containing trichloroacetic acid (10 % w/v). Zinc acetate (1 % w/v, 250 μl) was injected to trap the generated H₂S. A solution of N,N-dimethyl-p-phenylenediamine sulfate (20 μM; 133 μl) in 7.2 m HCl and FeCl₃ (30 μM; 133 μl) in 1.2 M HCl was added. Absorbance at 670 nm of the resulting mixture (300 μl) was determined after 10 min using a 96-well microplate reader (Bio-Rad). The H₂S concentration of each sample was calculated against a calibration curve of NaSH (1–500 μmol/ml). The protein concentration was measured with a BCA protein assay reagent kit (Pierce, Rockford, IL). The concentrations were expressed as micromole per gram protein.

The GSH level was assessed by the method of Hissin and Hilf [21] and Lee et al. [11]. This assay detects reduced GSH by its reaction with o-phthalaldehyde (OPT) at pH 8.0. Cells (10⁶) in 1.5-ml tubes were washed twice with PBS, and 200 μl of 6.5 % trichloroacetic acid (TCA) was added. The mixture was incubated on ice for 10 min and centrifuged (13,000 rpm, 1 min). The supernatant was discarded, and the pellets were resuspended in 200 μl of ice-cold 6.5 % TCA and centrifuged again (13,000 rpm, 2 min). Supernatants (7.5 μl) were transferred to 96-well plates containing 277.5 μl phosphate-EDTA buffer (pH 8.0) in 1 M NaOH solution. Then, 15 μl OPT (1 mg/ml in methanol) was added. The reaction mixture was incubated in the dark at room temperature for 25 min. The fluorescence at 350 nm excitation/420 nm emission was measured in a multiwell plate reader. The concentration was calculated from a standard curve using serial dilutions of reduced GSH. The concentration was expressed as micromole per gram protein.

Inflammatory stimulation of microglia or THP-1 cells causes them to release the inflammatory cytokines TNFα and IL-6 [22, 23]. Figure 3 shows the effect on TNFα release by treatment of glial cells with NaSH and STS (1–500 μM for 8 h pre-incubation, protocol 1). THP-1 release of TNFα (Fig. 3a) and IL-6 (Fig. 3b) and human microglial release of TNFα (Fig. 3c) and IL-6 (Fig. 3d) are illustrated. The release of TNFα (Fig. 3a) and IL-6 (Fig. 3b) was reduced by NaSH and STS exposure in a concentration-dependent manner (Fig. 3, NaSH: P < 0.01 for 1 μM or higher and for STS: P < 0.01 for 10 μM or higher). The inhibitory effects of NaSH were more powerful than STS (P < 0.01 for 1 μM or higher and for TNFα and P < 0.01 for 3 μM or higher for IL-6). The IC₅₀ values for NaSH and STS shown in Table 1 (A, B) were confirmatory. The pattern was similar in microglia (Fig. 3c, d). LPS/IFNγ stimulation caused a 9.5-fold increase of TNFα and an 11-fold increase of IL-6. Treatment with NaSH and STS reduced this release (NaSH: 75 % and STS: 50–60 % at 500 μM, P < 0.01).

For astrocytes, IL-6 is the main inflammatory mediator that is generated [24]. Figure 4 shows comparable data for IL-6 release from U373 cells (Fig. 4a) and cultured astrocytes (Fig. 4b). Cells were activated with IFNγ according to experimental protocol 1 and were then treated similarly to the THP-1 and microglial cells shown in Fig. 3. The release of IL-6 was more than fourfold higher in primary cultured astrocytes compared with U373 cells. However, both compounds reduced the release of IL-6 from IFNγ-activated U373 cells or astrocytes in a concentration-dependent manner (P < 0.01 NaSH: P < 0.01 from 1 μM and for STS: P < 0.01 from 10 μM). Again, NaSH is stronger in reducing IL-6 release in both cell types (P < 0.01 from 1 μM). The IC₅₀ values for NaSH and STS are shown in Table 1 (C). NaSH is more potent than STS in attenuating release of these cytokines.

We investigated, as described in methods, the direct effects of TNFα and IL-6 treatment, alone and in combination, on the viability of SH-SY5Y cells. MTT assays were performed after 3 days incubation at 37 °C. There were no changes in viability as shown in Fig. 5. However, the effects were different when the cells were exposed to cytokine-depleted CM from microglia and astrocytes. For depleted microglial conditioned medium (CM), the SH-SY5Y viability was increased by 10.6 % for TNFα, 8.7 % for IL-6, and 21 % for TNFα + IL-6 (Fig. 5a). For astrocytes, the corresponding increase in viability for IL-6 depletion was 22 % (Fig. 5b).

We investigated the effects of pre-treatment with NaSH and STS on the toxicity of CM toward SH-SY5Y cells. The CM was obtained by 2 days treatment of LPS/IFNγ-activated THP-1 cells and IFNγ-activated U373 cells. Experimental protocol 1 was followed.

It was found that NaSH and STS each attenuated SH-SY5Y cell viability loss by THP-1 CM (Fig. 6). The effect was concentration-dependent in the 1–500 μM range and was time-dependent up to 12 h (Fig. 6a: 2 h pre-incubation, 6b: 4 h pre-incubation, 6c: 8 h pre-incubation and 6d: 12 h pre-incubation). At the shortest time interval of 2 h and at the lowest concentration of 1 μM, the protective effect of NaSH was minimal. But the toxicity was reduced to about half at a concentration of 500 μM (P < 0.01 from 1 μM). By 12 h the protective effect had increased to the point where the lowest concentration reduced the toxicity by about one third while the highest concentration reduced it by about five- to sixfold. Pre-treatment with STS also attenuated THP-1 toxicity toward SH-SY5Y cells at 2 h pre-incubation (P < 0.01 from 50 μM) but was less effective than NaSH (P < 0.01 from 3 μM); protective levels were 75 % of those obtained with NaSH.

Figure 7 shows the effects of exposing SH-SY5Y cells to the neurotoxic CM from U373 cells that had been stimulated with 150 U of IFNγ according to experimental protocol 1. The figure shows highly similar data to those obtained from stimulated THP-1 cells (Fig. 7a: 2 h pre-incubation, 7b: 4 h pre-incubation, 7c: 8 h pre-incubation and 7d: 12 h pre-incubation) were obtained. Again, the protective effect of the both agents was both concentration-dependent and incubation time-dependent. NaSH was more neuroprotective than STS (MTT test data: P < 0.01 from 50 μM at 2 h, P < 0.01 from 30 μM at 4 h, P < 0.01 from 10 μM at 8 h, P < 0.01 from 3 μM at 12 h).

Table 2 summarized the IC₅₀ results of these studies. As shown in Figs. 6 and 7, the concentrations of NaSH required to reach the level of IC₅₀, for both THP-1 and U373 cells, are approximately threefold lower than those required for STS at all the incubation times in both THP-1 and U373 cells. The data indicate that IC₅₀s of NaSH and STS are reduced with a longer pre-incubation time. The IC₅₀s of NaSH were at least three times lower than those of STS in each pre-incubation time in both cell types.

We also investigated the effect of supernatants from LPS/IFNγ-stimulated microglia and IFNγ-stimulated astrocytes on SH-SY5Y cell viability after treatment with all the agents. Due to the limited availability of microglia and astrocytes, they were pre-exposed to the compounds at only one time period (8 h) prior to treatment with stimulatory agents (experimental protocol 1). For measuring SH-SY5Y cell viability, the MTT and LDH release assays were utilized (upper panel: MTT assays and lower panel: LDH release assays). It was observed that both NaSH and STS reduced the toxicity of both LPS/IFNγ-stimulated microglia (Fig. 8a) and IFNγ-stimulated astrocytes (Fig. 8b) toward SH-SY5Y cells (MTT assay data: for NaSH: P < 0.01 from 1 μM and for STS: P < 0.01 from 10 μM). Again, NaSH had a greater neuroprotective effect than STS (MTT assay data: P < 0.01 from 1 μM) (Table 3). These data demonstrate that the effects observed with cultured microglia and astrocytes are comparable to those observed with the THP-1 and U373 cell lines.

However, pre-treatment with NaSH or STS of THP-1 cells and U373 cells for 12 h and of microglia and astrocytes for 8 h with both compounds did not change the viability of any glial cells by stimulants (LPS/IFNγ for THP-1 cells and microglia: Additional file 1: Figure S1A and S1C, and IFNγ for U373 cells and strocytes: Additional file 1: Figure S1B and S1D).

It was also found that NaSH and STS were not directly protective of SH-SY5Y cells (Additional file 1: Figure S2). When they were added to the CM after stimulation had taken place (protocol 2, A: THP-1 cells and B: U373 cells), they had no effect. As the figure shows, there was no difference between the agents and there was no effect of concentration. This establishes that the agents were working by inhibiting the glial inflammatory response.

In summary, NaSH and STS were not toxic to the glial cells in the presence of inflammatory stimuli. The viability of SH-SY5Y cells treated with the CM from LPS/IFNγ-stimulated THP-1 and IFNγ-stimulated U373 cells was unchanged.

As further control experiments, we tested the effects of NaSH and STS on the growth and survival of human microglia and astrocytes under normal and inflammatory conditions. Human microglia and astrocytes were treated with NaSH or STS for 3 days. MTT assays were then performed on the cultures. Microglia and astrocytes were next treated with stimulants plus NaSH or STS for 2 days. The stimulants were LPS/IFNγ for microglia and IFNγ for astrocytes. Comparative MTT assays were then performed on the cultures. The results are shown in Additional file 1: Figure S3. It was found that STS and NaSH did not change the viability of microglia and astrocytes under any of the experimental conditions.

In the final set of experiments, we investigated the effects of pre-treatment with NaSH and STS at 100 μM on phospho-P38 MAPK and phospho-NFκB proteins. These are indicative of intracellular inflammatory activation. The data are shown in Fig. 9. Upon exposure to the stimulants, both microglia and astrocytes showed an increase in these proteins. For microglia, P38 MAPK was increased sevenfold and NFκB eightfold. For astrocytes, P38 MAPK and NFκB were increased sevenfold. Both compounds attenuated these increases, and NaSH was again more powerful than STS (NaSH: 80 % reduction and STS: 60–65 % reduction).