Dimethylfumarate inhibits microglial and astrocytic inflammation by suppressing the synthesis of nitric oxide, IL-1beta, TNF-alpha and IL-6 in an in-vitro model of brain inflammation

Abstract

Background: Brain inflammation plays a central role in multiple sclerosis (MS). Dimethylfumarate (DMF), the main ingredient of an oral formulation of fumaric acid esters with proven therapeutic efficacy in psoriasis, has recently been found to ameliorate the course of relapsing-remitting MS. Glial cells are the effector cells of neuroinflammation; however, little is known of the effect of DMF on microglia and astrocytes. The purpose of this study was to use an established in vitro model of brain inflammation to determine if DMF modulates the release of neurotoxic molecules from microglia and astrocytes, thus inhibiting glial inflammation.

Methods: Primary microglial and astrocytic cell cultures were prepared from cerebral cortices of neonatal rats. The control cells were treated with LPS, an accepted inducer of pro-inflammatory properties in glial cells, and the experimental groups with LPS and DMF in different concentrations. After stimulation/incubation, the generation of nitric oxide (NO) in the cell culture supernatants was determined by measuring nitrite accumulation in the medium using Griess reagent. After 6 hours of treatment RT-PCR was used to determine transcription levels of iNOS, IL-1beta, IL-6 and TNF-alpha mRNA in microglial and astrocytic cell cultures initially treated with DMF, followed after 30 min by LPS treatment. Moreover, we investigated possible involvement of the ERK and Nrf-2 transduction pathway in microglia using western blot analysis.

Results: Pretreatment with DMF decreased synthesis of the proinflammatory mediators iNOS, TNF-alpha, IL-1beta and IL-6 at the RNA level in activated microglia and astrocytes in vitro, associated with a decrease in ERK phosphorylation in microglia.

Conclusions: Collectively, these results suggest that the neuroprotective effects of DMF may be in part functionally attributable to the compound's ability to inhibit expression of multiple neuroinflammatory mediators in brain of MS patients.

Figure 1

Dimethyl fumarate inhibits LPS-induced mRNA expression of NO (A), iNOS, Il-1β, Il-6 and TNF-α (B), respectively, in cultivated microglia. LPS was added to microglia cells 30 min after the addition of dimethyl fumarate (10 μM) to analyze its effect on mRNA expression after 6 hours of treatment. mRNA expression was analyzed using TaqMan real-time RT-PCR and results for cells treated with dimethyl fumarate were compared to those of cells stimulated solely by LPS. 18s RNA (a housekeeping gene) was used as an internal control. The data were assessed from 3 independent experiments, each run in triplicate. Asterisks (* = p < 0.05; *** = p < 0.001), indicate significant differences compared to cells stimulated solely by LPS (ANOVA followed by the Bonferroni test).

Figure 2

Dimethyl fumarate inhibits LPS-induced mRNA expression of iNOS (A), Il-1β (B), Il-6 (C) and TNF-α (D) in astrocytes. LPS was added to astrocytes 30 min after the addition of dimethylfumarate (10 μM), its effect on mRNA expression was analyzed after 6 hours of treatment. mRNA expression was analyzed using TaqMan real-time RT-PCR and results for cells treated with dimethyl fumarate were compared to those of cells stimulated solely by LPS. 18s RNA (a housekeeping gene) was used as an internal control. The data were assessed from 3 independent experiments, each run in triplicate. Asterisks (* = p < 0.05; ** = p < 0,01), indicate significant differences compared to cells stimulated solely by LPS (ANOVA followed by the Bonferroni test). Note that the reduction of iNOS mRNA (Fig. 3A) is not statistically significant.

Figure 3

DMF inhibition of ERK phosphorylation in LPS-activated microglia. Results are compared to microglial cultures stimulated solely by LPS. Results are detected by western blot after 5 minutes of treatment. Activated ERK (phospho-ERK) species were detected by immunoblot analysis with antibodies specific for the phosphorylated forms of the kinase (A). The amount of protein loaded in each lane was confirmed by measurement of the amount of ERK using antibody against the unphosphorylated form of ERK. (B) Blots were subjected to densitometry and normalized to the signal of the respective pan-MAPK antibody. Results are representative of 3 independent experiments (*** = p < 0.001).

Figure 4

DMF-induced expression of Nrf2 protein in microglia. One hour after stimulation of microglia with DMF, nuclear proteins were isolated and separated using SDS-polyacrylamide gel electrophoresis, western blotted and probed with anti-Nrf2, and reprobed with anti-Lamin A/C (as a loading control) antibodies.