Novel disease-modifying anti-rheumatic drug iguratimod suppresses chronic experimental autoimmune encephalomyelitis by down-regulating activation of macrophages/microglia through an NF-κB pathway

Abstract

We aimed to elucidate the effects of iguratimod, a widely used anti-rheumatic drug with no severe side effects, on chronic experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Iguratimod was orally administered to mice immunised with myelin oligodendrocyte glycoprotein peptide 35-55. Preventive administration of iguratimod from the time of immunisation was found to markedly reduce the clinical severity of acute and chronic EAE. Pathologically, iguratimod treatment significantly reduced demyelination and infiltration of CD3⁺ T, F4/80⁺, and CD169⁺ cells into the spinal cord, and suppressed macrophage/microglia activation in the parenchyma at the acute and chronic stages compared with vehicle treatment. Therapeutic administration of iguratimod after the onset of clinical symptoms significantly ameliorated the clinical severity of chronic EAE and reduced demyelination, T helper (Th)1/Th17 cell infiltration, macrophage/microglia activation, and nuclear factor (NF)-κB p65 and cyclooxygenase-2 expression in the spinal cord. In vitro, iguratimod treatment inhibited nuclear translocation of NF-κB p65 and down-regulated pro-inflammatory responses in macrophages and microglia. Our results suggest that iguratimod ameliorates acute and chronic EAE by suppressing inflammatory cell infiltration and immune cell activation, partly through inhibition of NF-κB p65, supporting the therapeutic potential of this drug for not only acute, but also chronic MS.

Figure 1

Iguratimod treatment prevents the development of MOG_35–55-induced EAE in C57BL/6 mice and inhibits its related demyelination. Oral iguratimod was administered daily started from the day of immunisation. (a) EAE clinical scores (means ± SEM) over time in mice treated with iguratimod (filled squares, n = 14) or CMC vehicle (filled circles, n = 15). Horizontal bars in the graph show the periods of days with significant differences between the two groups. The lower table shows the following parameters as means ± SEM: incidence, day of onset, and maximum clinical score of each group. *p < 0.05, CMC vehicle-treated mice versus iguratimod-treated mice. (b) Cryostat cross-sections (20-µm thickness) of the lumbar spinal cord of iguratimod-treated mice and vehicle-treated mice. DAPI is shown in blue and myelin staining is shown in red. Scale bar: 500 µm. (c) Quantification of myelin density in the anterior white matter at day 15 (n = 4 per group) and day 50 (n = 5 per group) (identified by myelin staining). (d) Quantification of myelin density in posterior parts of the white matter at day 15 (n = 4 per group) and day 50 (n = 5 per group) (identified by myelin staining).

Figure 2

Preventive iguratimod administration suppresses inflammatory cell infiltration and microglial activation in MOG_35–55-induced EAE. (a) Iguratimod treatment suppresses DAPI⁺CD3⁺ cell infiltration in the lumbar spinal cord of EAE mice. Scale bar: 100 μm. (b) Areas of DAPI⁺CD3⁺ cells quantified at day 15 (n = 4 per group) and day 50 (n = 5 per group). (c) Iguratimod treatment suppresses DAPI⁺F4/80⁺ cell infiltration in the lumbar spinal cord of EAE mice. Scale bar: 100 μm. (d) Areas of DAPI⁺F4/80⁺ cells quantified at day 15 (n = 4 per group) and day 50 (n = 5 per group). (e) Iguratimod treatment suppresses Iba-1 up-regulation in the lumbar spinal cord of mice with EAE. Scale bar: 100 μm. (f) Areas of DAPI⁺Iba-1⁺ cells quantified at day 15 (n = 4 per group) and day 50 (n = 5 per group). (g) Microglial cell circularity quantified at day 15 (n = 4 per group) and day 50 (n = 5 per group). (h) Iguratimod treatment suppresses DAPI⁺CD169⁺ cell infiltration in the lumbar spinal cord of EAE mice. Scale bar: 100 μm. (i) Iguratimod treatment suppresses DAPI⁺CD11b⁺ cell infiltration in the lumbar spinal cord of EAE mice. Scale bar: 100 μm. (j) Areas of DAPI⁺CD169⁺ cells quantified at day 15 (n = 4 per group). (k) Areas of CD11b⁺ cells quantified at day 15 (n = 4 per group). *p < 0.05, **p < 0.01. Error bars indicate SEM. An unpaired Student’s t-test was used for analysis.

Figure 3

Therapeutic administration of iguratimod improves the severity of chronic EAE by suppressing inflammatory cell infiltration and microglia activation. Oral iguratimod was administered daily starting on the day of clinical onset (10 days after immunisation). (a) EAE clinical scores (means ± SEM) and body weights (means ± SEM) over time in mice treated with iguratimod (filled squares, n = 6) or CMC vehicle (filled circles, n = 11). Horizontal bars in the graphs show the periods of days with significant differences between the two groups. (b) Cryostat cross-sections (20 µm thickness) of the lumbar spinal cord of iguratimod-treated mice and vehicle-treated mice. DAPI is shown in blue and myelin staining is shown in red. Scale bar: 500 µm. (c) Quantification of myelin density identified by myelin staining in the anterior or posterior parts of the white matter at day 50 (n = 11 for control group; n = 6 for IGU group). (d) Iguratimod treatment after the onset of EAE suppresses DAPI⁺CD45⁺ and DAPI⁺CD3⁺ cell infiltration and decreases Iba-1 fluorescence in the lumbar spinal cord of mice with chronic EAE at day 50. Scale bar: 100 µm. (e) Iguratimod treatment after the onset of EAE decreases DAPI⁺F4/80⁺ and DAPI⁺CD11b⁺ cells and NOS2 fluorescence, but does not change arginase-1 fluorescence in the lumbar spinal cord of mice with chronic EAE at day 50. Scale bar: 100 µm. (f) Areas of CD45⁺ cells quantified at day 50 (n = 11 for control group, n = 6 for IGU group). (g) Areas of CD3⁺ cells quantified at day 50 (n = 11 for control group; n = 6 for IGU group). (h) Areas of Iba-1⁺ cells quantified at day 50 (n = 11 for control group; n = 6 for IGU group). (i) Microglial cell circularity (n = 11 for control group; n = 6 for IGU group). (j) Areas of CD11b⁺ cells quantified at day 50 (n = 11 for control group; n = 6 for IGU group). (k) Areas of F4/80⁺ cells quantified at day 50 (n = 11 for control group; n = 6 for IGU group). (l) NOS2 signal intensity quantified at day 50 (n = 11 for control group; n = 6 for IGU group). (m) Arginase-1 signal intensity quantified at day 50 (n = 11 for control group; n = 6 for IGU group). (n) Iguratimod treatment after the onset of EAE suppresses DAPI⁺/CD169⁺ cell infiltration in the lumbar spinal cord of mice with chronic EAE at day 50. (o) Areas of CD169⁺ cells quantified at day 5 (n = 11 for control group; n = 6 for IGU group). *p < 0.05, **p < 0.01, ***p < 0.0001. Error bars indicate SEM. An unpaired Student’s t-test was used for analyses.

Figure 4

Therapeutic administration of iguratimod suppresses COX-2 and NF-κB p65 expression in the EAE spinal cord. (a) Iguratimod treatment after the onset of EAE decreases COX-2 fluorescence intensity. (b) COX-2 signal intensity was quantified at day 50 (n = 11 for control group; n = 6 for IGU group). (c) Iguratimod treatment after the onset of EAE decreases NF-κB p65 fluorescence intensity. (d) NF-κB p65 fluorescence intensity was quantified at day 50 (n = 11 for control group; n = 6 for IGU group). Scale bar: 100 µm. *p < 0.05. Error bars indicate SEM. An unpaired Student’s t-test was used for analyses.

Figure 5

Flow cytometric analysis of macrophages and T cells in iguratimod-treated chronic EAE mice. (a) CD45⁺CD4⁺ T cells and CD45⁺F480⁺ macrophages in CNS tissues (brain and spinal cord) from one representative control or IGU-treated EAE mouse. Numbers on plots are percentages of double-positive cells among gated viable cells. Iguratimod decreases CD45⁺CD4⁺ T cells and CD45⁺F480⁺ macrophages in EAE CNS tissues. (b) Bar graphs showing percentages of CD45⁺CD4⁺ T cells and CD45⁺F480⁺ macrophages in CNS tissues (brain and spinal cord) from EAE mice. Data are summarised for 5 mice per group. (c) IFN-γ⁺CD4⁺ Th1 cells and IL-17A⁺CD4⁺ Th17 cells in CNS tissues (brain and spinal cord) from one representative control or IGU-treated EAE mouse. Numbers on plots are percentages of double-positive cells among MHC-II⁻CD4⁺ gated cells. Iguratimod decreases IFN-γ⁺CD4⁺ Th1 and IL-17A⁺CD4⁺ Th17 cell percentages among T cells infiltrating the EAE CNS. T cells were stimulated with PMA/ionomycin for 5 h. (d) Bar graphs showing percentages of IFN-γ⁺CD4⁺ Th1 cells and IL-17A⁺CD4⁺ Th17 cells among CD4⁺ T cells infiltrating the CNS tissues (brain and spinal cord) from EAE mice. Data are summarised for 5 mice per group. (e) IFN-γ⁺CD4⁺ Th1 cells and IL-17A⁺CD4⁺ Th17 cells in the spleen of one representative control or IGU-treated EAE mouse. Numbers on plots are percentages of double-positive cells among MHC-II⁻CD4⁺ gated cells. Iguratimod decreases IFN-γ⁺CD4⁺ Th1 and IL-17A⁺CD4⁺ Th17 cell percentages in the EAE spleen. T cells were stimulated with PMA/ionomycin for 5 h. (f) Bar graphs showing percentages of IFN-γ⁺CD4⁺ Th1 cells and IL-17A⁺CD4⁺ Th17 cells in the spleen of EAE mice. Data are summarised for 5 mice per group. (g) CD25⁺FoxP3⁺ Treg cells in the spleen of one representative control or IGU-treated EAE mouse. Numbers on plots are percentages of CD4⁺ gated cells. Iguratimod does not significantly change CD25⁺FoxP3⁺ Treg cell percentages in the EAE spleen. (h) Bar graphs showing percentages of CD25⁺FoxP3⁺ Treg cells in the spleen of EAE mice. Data are summarised for 5 mice per group. **p < 0.01, ***p < 0.0001, N.S. not significant. Error bars indicate SEM. An unpaired Student’s t-test was used for analyses.

Figure 6

Iguratimod down-regulates the increased F4/80 and NOS2 expression in macrophages after LPS stimulation in vitro. (a) Representative images of F4/80 (red) and NOS2 (green) immunofluorescence in macrophages after stimulation with DMSO vehicle (control), LPS (10 ng/ml), or LPS (10 ng/ml) plus iguratimod (30 µg/ml) for 2 h. Cell nuclei were detected by DAPI (blue). Scale bar: 100 µm. (b) Quantification of F4/80 (n = 7 for control; n = 8 for LPS; n = 8 for LPS + IGU) and NOS2 (n = 11 for control; n = 11 for LPS; n = 10 for LPS + IGU) signal intensity in macrophages. (c) Protein levels of NOS2 in macrophages determined by western blotting after stimulation with DMSO vehicle (control), LPS (10 ng/ml), or LPS (10 ng/ml) plus iguratimod (30 µg/ml) for 24 h (n = 3 per group). Full length blots are presented in Supplementary Figure S2. (d) Representative images of NF-κB p65 immunofluorescence (red) in macrophages after stimulation with DMSO vehicle (control), LPS (10 ng/ml), or LPS (10 ng/ml) plus iguratimod (30 µg/ml) for 2 h. Cell nuclei were detected by DAPI (blue). Scale bar: 20 µm. (e) Quantification of NF-κB p65 signal intensity in macrophages (n = 6 for control; n = 5 for LPS; n = 6 for LPS + IGU). *p < 0.05, **p < 0.01, ***p < 0.0001. Error bars indicate SEM. One-way ANOVA with Tukey’s post hoc test was used for analyses.

Figure 7

Iguratimod inhibits the up-regulation of NF-κB p65, Iba-1, NOS2, and CD11b in microglia after LPS stimulation in vitro. (a) Representative images of Iba-1 (red) and NF-κB p65 (green) immunofluorescence in microglia after stimulation with DMSO vehicle (control), LPS (10 ng/ml), or LPS (10 ng/ml) plus iguratimod (30 µg/ml) for 2 h. Cell nuclei were detected by DAPI (blue). Scale bar: 20 µm. (b) Representative images of NOS2 (red) and CD11b (green) immunofluorescence in microglia after stimulation with DMSO vehicle (control), LPS (10 ng/ml), or LPS (10 ng/ml) plus iguratimod (30 µg/ml) for 2 h. Cell nuclei were detected by DAPI (blue). Scale bar: 20 µm. (c) Quantification of NF-κB p65 (n = 5 for control; n = 6 for LPS; n = 7 for LPS + IGU), Iba-1 (n = 6 for control; n = 5 for LPS; n = 5 for LPS + IGU), NOS2 (n = 6 for control = 6, n = 6 for LPS; n = 4 for LPS + IGU), and CD11b (n = 6 for control; n = 5 for LPS; n = 5 for LPS + IGU) signal intensity in microglia. *p < 0.05, **p < 0.01, ***p < 0.0001. Error bars indicate SEM. One-way ANOVA with Tukey’s post hoc test was used for analyses.

Figure 8

Phenotypes of activated macrophages (M1/M2) after stimulation with or without iguratimod in vitro. (a) NF-κB p65 transcripts were quantified by real-time PCR. (b–g) M1 marker gene transcripts were quantified by real-time PCR. (b), NOS2; (c), IL-12; (d), CD80; (e), CD86; (f), IL-6; (g), IL-1b. (h–l) M2 marker gene transcripts were quantified by real-time PCR. (h), CXCR1; (i), IL-10; (j), CD163; (k), CD23; (l), arginase-1; Values were normalised by GAPDH, and the fold changes compared with GAPDH are shown (n = 3 per group). *p < 0.05, **p < 0.01, ***p < 0.0001. Error bars indicate SEM. One-way ANOVA with Tukey’s post hoc test was used for analyses.