Targeting the shift from M1 to M2 macrophages in experimental autoimmune encephalomyelitis mice treated with fasudil

Abstract

We observed the therapeutic effect of Fasudil and explored its mechanisms in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Fasudil, a selective Rho kinase (ROCK) inhibitor, was injected intraperitoneally at 40 mg/kg/d in early and late stages of EAE induction. Fasudil ameliorated the clinical severity of EAE at different stages, and decreased the expression of ROCK-II in spleen, accompanied by an improvement in demyelination and inhibition of inflammatory cells. Fasudil mainly inhibited CD4(+)IL-17(+) T cells in early treatment, but also elevated CD4(+)IL-10(+) regulatory T cells and IL-10 production in late treatment. The treatment of Fasudil shifted inflammatory M1 to anti-inflammatory M2 macrophages in both early and late treatment, being shown by inhibiting CD16/32, iNOS, IL-12, TLR4 and CD40 and increasing CD206, Arg-1, IL-10 and CD14 in spleen. By using Western blot and immunohistochemistry, iNOS and Arg-1, as two most specific markers for M1 and M2, was inhibited or induced in splenic macrophages and spinal cords of EAE mice treated with Fasudil. In vitro experiments also indicate that Fasudil shifts M1 to M2 phenotype, which does not require the participation or auxiliary of other cells. The polarization of M2 macrophages was associated with the decrease of inflammatory cytokine IL-1β, TNF-α and MCP-1. These results demonstrate that Fasudil has therapeutic potential in EAE possibly through inducing the polarization of M2 macrophages and inhibiting inflammatory responses.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Fasudil delays onset, and ameliorates severity of EAE.

Chronic EAE was induced in C57BL/6 mice with MOG35–55. Fasudil was injected (i.p.) at 40 mg/kg/d every other day on day 3 p.i. (Fasudil early treatment, n = 13) or on day 11 p.i. (Fasudil late treatment, n = 13). The horizontal line is treatment period of Fasudil. The injection of normal saline was set up as control (EAE control, n = 13) in a similar manner. a) Mean clinical score, b) Mean body weight. The comparison in each time point was separately analyzed by Mann Whitney U test after nonparametric Kruskal-Wallis test. *p<0.05; **p<0.01.

Figure 2. Fasudil inhibits inflammation and improves myelination in spinal cords.

Chronic EAE was induced in C57BL/6 mice with MOG35–55. Fasudil was injected (i.p.) at 40 mg/kg/d every other day on day 3 p.i. (Fasudil early treatment, n = 9) or on day 11 p.i. (Fasudil late treatment, n = 9). The injection of normal saline was set up as control (EAE control, n = 12) in a similar manner. On day 21 p.i., spinal cords were used for Luxol fast blue and H&E staining. Left) Demyelination stained with Luxol Fast Blue. Total white matter in Luxol Fast Blue was manually outlined, and pixel area (%) of demyelination in total white matter was calculated by Image-Pro Plus software. Right) Inflammation stained with H&E. Color photo in H&E stain was automatically converted to black and white, and number of inflammatory foci (>20 mononuclear cells/focus) in whole spinal cords were calculated by Image-Pro Plus software. Quantitative results are analyzed for 6 mice in each group and are representative of 3 experiments with similar results. *p<0.05, **p<0.01.

Figure 3. Fasudil inhibits the expression of ROCK-II in spleen.

Chronic EAE was induced in C57BL/6 mice with MOG35–55. Fasudil was injected (i.p.) at 40 mg/kg/d every other day on day 3 and 11 p.i. On day 21 p.i., spleens were collected, and the expression of ROCK II was measured by immunohistochemistry. Spleen from healthy mice was measured as control. Up) Immunohistochemistry. Green = ROCK-II; Blue = Hoechst nuclear staining. As a negative control, additional section was treated similarly, but the primary antibodies were omitted. Quantitative results are analyzed by Western blot. Down) Western blot. Left down = representative bands from one of three experiments with similar results; Right down = quantitative results are analyzed for 5 mice in each group and expressed as fold change relative to GAPDH as the loading control *p<0.05.

Figure 4. Fasudil induces the regulatory T cells and inhibits IL-17 T cells.

Chronic EAE was induced in C57BL/6 mice with MOG35–55. Fasudil was injected (i.p.) at 40 mg/kg/d every other day on days 3 and 11 p.i. Mice were sacrificed and splenic MNCs were prepared on days 9 (a) and 21 (b) p.i. Cells were stained with T cell marker CD4 and CD25, IL-10 and IL-17, and subsets of T cells were analyzed using flow cytometry. Scatterplot, representative dot plots from one of three experiments with similar results. Histogram, quantitative results are analyzed for 5 mice in each group. Results are expressed as the percentage of double positive cells in four-quadrant diagram or within rectangle. *p<0.05, **p<0.01, ***p<0.001.

Figure 5. Fasudil shifts M1 to M2 phenotype.

Chronic EAE was induced in C57BL/6 mice with MOG35–55. Fasudil was injected (i.p.) at 40 mg/kg/d every other day on day 3 and 11 p.i. Mice were sacrificed and splenic MNCs were prepared on day 9 (a) and 21 (b) p.i.. Cells were stained with macrophage marker F4/80 and M1/M2 markers, and the polarization of M1/M2 was analyzed using flow cytometry. Scatterplot, representative dot plots from one of three experiments with similar results. Histogram, quantitative results are analyzed for 5 mice in each group. Results are expressed as the percentage of double positive cells in four-quadrant diagram. *p<0.05, **p<0.01, ***p<0.001.

Figure 6. Fasudil enhances the ratio of Arg-1/iNOS.

Chronic EAE was induced in C57BL/6 mice with MOG35–55. Fasudil was injected (i.p.) at 40 mg/kg/d every other day on days 3 and 11 p.i. On day 21 p.i., mice were sacrificed, and protein was extracted from splenic macrophages and spinal cords. a) Representative bands of Western blot for iNOS and Arg-1 from one of two experiments with similar results. Quantitative analysis of iNOS and Arg-1 expression in splenic macrophages and spinal cords are analyzed for 4–5 mice in each group. Results are expressed as fold change relative to GAPDH as the loading control. *p<0.05, **p<0.01, ***p<0.001. b) The double staining of CD68 (green) and iNOS/Arg-1 (red) by immunohistochemistry. Representative patterns were obtained from one of three experiments with similar results.

Figure 7. Fasudil directly shifts M1 to M2 phenotype in macrophages.

Macrophages from healthy mice were purified and stimulated with IFN-γ. Fasudil was added for 12 h immediately after IFN-γ treatment. The levels of iNOS and Arg-1 were measured by Western blot (a) and flow cytometry (b). The results were analyzed from three independent experiments. *p<0.05, **p<0.01.

Figure 8. Fasudil inhibits inflammatory molecules.

Chronic EAE was induced in C57BL/6 mice with MOG35–55. Fasudil was injected (i.p.) at 40 mg/kg/d every other day on days 3 and 11 p.i. On day 21 p.i., spleen was obtained, and splenic MNCs were prepared. Splenic MNCs were incubated for 48 h at 37°C in the presence of MOG35–55 (10 µg/ml). Splenic MNCs from healthy mice were measured as control. The supernatants were collected, and the levels of IL-10, IL-17 (a), IL-1β, IL-6, TNF-α and MCP-1 (b) were measured by ELISA kits. The results were represented as pg/ml, and analyzed from 5 mice in each group. *p<0.05, **p<0.01.