Fumaric acid esters are effective in chronic experimental autoimmune encephalomyelitis and suppress macrophage infiltration

Abstract

Fumaric acid esters (FAE) have proven their therapeutic efficacy in psoriasis, a Th1 mediated skin disease. More recently, preliminary data have suggested an activity in multiple sclerosis (MS) as well. To investigate further possible mechanisms of action of these compounds in inflammatory diseases, we studied the FAE methyl hydrogen fumarate (MHF) and dimethyl fumarate (DMF) in chronic experimental autoimmune encephalomyelitis (EAE) induced by immunization of C57BL/6 mice with MOG peptide aa 35-55. Preventive treatment with these FAE was delivered twice a day by oral gavage. Both esters had a significant therapeutic effect on the disease course and histology showed a strongly reduced macrophage inflammation in the spinal cord. Multiparameter cytokine analysis from blood detected an increase of IL-10 in the treated animals. We conclude that the underlying biological activity of FAE in EAE is complex and, to elucidate the molecular mechanisms, further investigation is needed.

Fig. 1

(a) Mean EAE disease scores ± standard error of the mean (SEM) of C57BL/6 mice treated with DMF 5 mg/kg (upright triangle), DMF 15 mg/kg (inverted triangle), MHF (diamond) and carrier alone (square). Preventive treatment was started on day 3. The treatment effect was significant for MHF (P < 0·0001 compared to the control group, two-way anova) and also for DMF, with the 15 mg/kg dose being more effective (P < 0·0001) than the 5 mg/kg dose (P < 0·001). (b) Mean weight and SEM of C57BL/6 mice which were treated with DMF 5 mg/kg (upright triangle), DMF 15 mg/kg (inverted triangle), MHF (diamond) and carrier alone (square) in the experiment shown in (a). DMF 5 mg/kg does not differ much from the control group, whereas the DMF 15 mg/kg group already exhibits lower weight in the beginning. MHF treated animals loose significantly less weight than the control animals (P < 0·001, two-way anova).

Fig. 2

Representative areas from spinal cord cross sections show reduced infiltration of macrophages (a,c) and T cells (b,d) in control animals (a,b) compared to MHF treated animals (c,d) after immunohistochemistry. Nuclei were counterstained with haematoxylin, 5 µm sections.

Fig. 3

Blinded quantification of inflammation in spinal cord during chronic EAE (27 days p.i). Control n = 11, 5 mg/kg DMF n = 11, 15 mg/kg DMF n = 11, MHF n = 12. Bars show mean cell count per mm². (a) The infiltration of CD3 + cells (T cells) was nonsignificantly reduced with DMF and significantly reduced with MHF (P < 0·01, Mann–Whitney U-test). (b) The infiltration of Mac-3+ cells (macrophages) was reduced nonsignificantly with 5 mg/kg DMF, significantly with 15 mg/kg DMF (P < 0·01, Mann–Whitney U-test) and highly significantly reduced with MHF (P < 0·001).

Fig. 4

For intrasample validation of cytokine measurement by MAP we divided aliquots of plasma samples from 5 healthy animals. Bars represent aliquots of the same sample. Baseline values of TNF-α (1 tick = 1 ng/ml) were under the detection limit. SEM for IL-10 (1 tick = 100 pg/ml) and IL-5 (1 tick = 100 pg/ml) was low, whereas it was rather high for IFN-γ (1 tick = 1 pg/ml).

Fig. 5

IL-5 levels during EAE with FAE therapy by MAP-analysis.

Fig. 6

IL-10 levels during EAE with FAE therapy by MAP-analysis. IL-10 was non-significantly elevated in the FAE treated groups. Shown are mean ± SEM.