The anti-inflammatory effects of dimethyl fumarate in astrocytes involve glutathione and haem oxygenase-1

Abstract

DMF (dimethyl fumarate) exerts anti-inflammatory and pro-metabolic effects in a variety of cell types, and a formulation (BG-12) is being evaluated for monotherapy in multiple sclerosis patients. DMF modifies glutathione (GSH) levels that can induce expression of the anti-inflammatory protein HO-1 (haem oxygenase-1). In primary astrocytes and C6 glioma cells, BG-12 dose-dependently suppressed nitrite production induced by either LI [LPS (lipopolysaccharide) at 1 μg/ml plus IFNγ (interferon γ) at 20 units/ml] or a mixture of pro-inflammatory cytokines, with greater efficacy in C6 cells. BG-12 reduced NOS2 (nitric oxide synthase 2) mRNA levels and activation of a NOS2 promoter, reduced nuclear levels of NF-κB (nuclear factor κB) p65 subunit and attenuated loss of IκBα (inhibitory κBα) in both cell types, although with greater effects in astrocytes. In astrocytes, LI decreased mRNA levels for GSHr (GSH reductase) and GCL (c-glutamylcysteine synthetase), and slightly suppressed GSHs (GSH synthetase) mRNAs. Co-treatment with BG-12 prevented those decreased and increased levels above control values. In contrast, LI reduced GSHp (GSH peroxidase) and GCL in C6 cells, and BG-12 had no effect on those levels. BG-12 increased nuclear levels of Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2), an inducer of GSH-related enzymes, in astrocytes but not C6 cells. In astrocytes, GSH was decreased by BG-12 at 2 h and increased at 24 h. Prior depletion of GSH using buthionine-sulfoximine increased the ability of BG-12 to reduce nitrites. In astrocytes, BG-12 increased HO-1 mRNA levels and effects on nitrite levels were blocked by an HO-1 inhibitor. These results demonstrate that BG-12 suppresses inflammatory activation in astrocytes and C6 glioma cells, but with distinct mechanisms, different dependence on GSH and different effects on transcription factor activation.

Figure 1. BG-12 reduces nitrite production

(A) Primary rat astrocytes were treated with LI to induce NOS2 expression, in the presence of the indicated concentration of BG-12 or vehicle (20 μM DMSO). After 24 h, nitrite levels in the media were measured by Griess assay. Results are means±S.D. of n = 6 per group and show % nitrite release as compared with LPS/IFNγ alone (none). The experiment was repeated twice with similar results. *P<0.001 versus vehicle, 1-way ANOVA, Bonferroni post hoc. (B) Measurements of LDH release after 24 h did not reveal any significant increase due to incubation with BG-12 up to 20 μM. (C) Rat C6 cells were treated with LI to induce NOS2 expression, in the presence of the indicated concentration of BG-12 or vehicle (20 μM DMSO). After 24 h nitrite levels in the media were measured by Griess assay. Results are means±S.D. of n = 6 per group and show % nitrite release as compared with LPS/IFNγ alone (none), which was 8.1 nmol of nitrite per mg per 24 h. The experiment was repeated twice with similar results. *P<0.001 versus vehicle, one-way ANOVA, Bonferroni post hoc. (D) Measurement of LDH release after 24 h did not reveal any significant increase due to incubation with BG-12 up to 20 μM.

Figure 2. Effects of BG-12 on cytokine-induced NOS2 expression

(A) Astrocytes and (B) C6 cells were treated with TII (a mixture of TNFα, IL-1β and IFNγ) to induce NOS2 expression, in the presence of the indicated concentration of BG-12 or vehicle (20 μM DMSO). After 24 h, nitrite levels in the media were measured by Griess assay. Results are means±S.D. of n = 4 per group and show % nitrite release as compared with TII alone (0 BG-12), which was 16 nmol (for astrocytes) and 46 nmol (for C6 cells) nitrite per mg per 24 h. The experiment was repeated twice with similar results. *P<0.001 versus vehicle, one-way ANOVA, Bonferroni post hoc.

Figure 3. BG-12 reduces NOS2 expression

(A) Primary astrocytes and (B) C6 cells were treated with LPS/IFNγ for 6 h alone, or in the presence of 10 μM BG-12 or equivalent amount of DMSO (Veh), then RNA samples were measured by qPCR for NOS2 mRNA levels. Results show the relative NOS2 mRNA level after normalization to β-tubulin mRNA values measured in the same samples. Results are means±S.D. for three samples from each group. LPS/IFNγ increased NOS2 mRNA levels approx. 2600-fold in astrocytes, and 1800-fold in C6 cells over baseline levels. *P<0.05 versus vehicle. (C) C6 cells stably transfected with rat 2.2 kb NOS2 promoter driving luciferase activity were treated with LPS/IFNγ in the presence of the indicated concentrations of BG-12 or vehicle (20 μM DMSO). After 5 h, luciferase activity was measured. Results are means±S.D. for three to six samples per group and show promoter activation relative to no BG-12. Treatment with LPS/IFNγ increased the NOS2 promoter 65-fold over control levels. Experiments were repeated twice with similar results. *P<0.05 versus vehicle, one-way ANOVA, Bonferroni post hoc.

Figure 4. BG-12 reduces NF-κB activation

(A) Astrocytes and C6 cells were treated with LPS/IFNγ in the presence of 20 μM BG-12 (open circles) or vehicle (filled circles) and after the indicated times nuclear lysates prepared and levels of active nuclear p65 binding to DNA measured with the TransAM (p65) assay kit. The amount of active p65 was normalized to levels measured in the non-treated (zero time) samples. Results are means±S.E., n = 3 or 4 per group. BG-12 significantly reduced p65-binding activity in both cells types (F[3,1] =  6.7 in astrocytes, 7.1 in C6 cells; P<0.005, two-way ANOVA; *P<0.05; ***P<0.001, Bonferroni post hoc). (B) Astrocytes and C6 cells grown on glass coverslips were treated as described above then fixed and stained for the presence of NF-κB p65. After 1 h incubation, nuclear staining for p65 was observed in most of the vehicle-treated cells, was absent from the BG-12-treated astrocytes and was still present in many of the BG-12-treated C6 cells (arrowheads).

Figure 5. Effects of BG-12 on IκBα levels

(A) Astrocytes and (B) C6 cells were treated with LPS/IFNγ in the presence of 20 μM BG-12 (‘BG’) or vehicle (‘Veh’), and after indicated times whole cell lysates prepared and used for Western-blot analysis of IκBα protein. The membranes were stripped and re-probed for levels of β-actin. (C) Band intensities were determined using ImageJ. Results are means±S.E. ratio IκBα to β-actin, n = 3–6 samples per group. BG-12 significantly reduced IκBα loss in both cell types (P<0.005, two-way ANOVA; *P<0.05, ***P<0.0001 versus BG-12, Bonferroni post hoc).

Figure 6. Effects of BG-12 on GSHs, GSHp, GSHr and GCL mRNA levels

Primary astrocytes (A) or C6 glioma cells (B) were treated with nothing (control), LPS/IFNγ or LPS/IFNγ in the presence of 10 μM BG-12 or equivalent amount of DMSO vehicle. After 6 h, the relative levels of the indicated mRNAs were determined by qPCR. Results show expression levels after normalization to β-tubulin mRNA values measured in the same samples, and relative to levels measured in the control samples. Results are means±S.D. for three samples per group. Results were analysed by one-way ANOVA and Bonferroni post hoc; *P<0.05 versus LI alone; #P<0.05 versus vehicle; §P<0.05 versus control.

Figure 7. BG-12 induces Nrf2 activation in astrocytes

(A) Astrocytes and (B) C6 cells were treated with LPS/IFNγ in the presence of 20 μM BG-12 (open circles) or vehicle (filled circles), and after the indicated times the nuclear lysates were prepared and levels of active nuclear Nrf2 binding to DNA measured with the TransAM Nrf2 assay kit. The amount of active Nrf2 was normalized to levels measured in the non-treated (zero time) samples. Results are means±S.E., n = 3 or 4. BG-12 significantly increased Nrf2-binding activity in astrocytes (F[3,1] = 3.6, P = 0.031; two-way ANOVA; *P<0.05 versus BG-12, Bonferroni post hoc) but had no effect in C6 cells.

Figure 8. Effects of BG-12 on GSH levels

(A) Primary astrocytes were treated with LPS/IFNγ together with BG-12 or vehicle at the indicated doses and for either 2 or 24 h, after which intracellular total GSH levels were measured. (B) C6 cells were incubated with LPS/IFNγ in the presence of the indicated concentration of BG-12 or vehicle. After 24 h, levels of free GSH were measured in whole cell lysates. Results show the GSH level relative to control value measured after 2 h in 3 μM vehicle, and are means±S.D. for three samples per group. *P<0.05 versus vehicle.

Figure 9. GSH depletion increases inhibition by BG-12

Primary astrocytes and C6 cells were pretreated with 500 μM BSO overnight to deplete GSH levels. The next day the cells were treated with LPS/IFNγ together with the indicated doses of BG-12 or vehicle, and nitrite levels were measured 24 h later. Results are means±S.D. for three samples per group, and shows the % nitrite production measured in the presence of BG-12 versus that measured in the equivalent amount of vehicle. *P<0.05 versus no BSO pretreatment.

Figure 10. BG-12 induces HO-1 expression in astrocytes

Primary astrocytes (A) or C6 glioma cells (B) were treated with nothing (C, open bars) or with LPS/IFNγ (filled bars) alone, with 10 μM BG-12 or with an equivalent amount of vehicle. After 6 h, relative levels of HO-1 mRNA were determined by qPCR. Resuls are means±S.D. of three samples per group of mRNA levels after normalization to β-tubulin mRNA values measured in the same samples, and is fold-increase relative to levels in astrocyte control samples. *P<0.05 versus vehicle.

Figure 11. Effects of BG-12 in astrocytes involve HO-1 activity

Primary astrocytes (A) or C6 glioma cells (B) were treated with LPS/IFNγ in the presence of indicated concentration of BG-12, and either 0 (open bars) or 1 μM (filled bars) ZnPP to block HO-1 activity. Nitrite levels were measured after 24 h. Results are means±S.D. for three samples per group and are nitrite production relative to that measured in the absence of BG-12 or ZnPP. *P<0.05 versus no ZnPP.