Pathological and inflammatory cytokine changes in EAE
The main pathological changes in MS are inflammation and demyelination in the white matter of the CNS, axonal injury and axonal loss, which can coexist and are often associated with diffuse lesions of the CNS[21]. As shown in Figure1c, a small number of monocyte and lymphocyte infiltrations could be seen in the spinal cord of EAE mice, with obvious perivascular inflammatory infiltrations by HE stainings. LFB stainings showed demyelination in the spinal cord.
The pro-inflammatory cytokines, IL-17A, IL-6, TNF-α, IL-12p70 and IL-1ß increased at the disease onset(P<0.0001) and IL-17A, IL-6, TNF-α, IL-12p70 decrease at the peak hour(P<0.0001). While anti-inflammatory cytokines, IL-4 and IL-10, significantly increased during the peak period(P<0.001)(Figure1d).
Clinical course, pathological and inflammatory cytokine changes after M2 macrophage treatment
The proportion of M2 macrophages cultured in vitro was more than 97% determined by flow cytology(Figure 2a). Those were transfused into the mice when EAE mice showed clinical symptoms on day 10 p.i.(onset). EAE treated with M2 macrophage(M2-treated) and PBS developed the disease at the same time; however, the peak symptoms were lighter and recovered earlier in the M2-treated group than controls. Similarly, weight records of the two groups showed that the weight gradually decreased in the EAE control group until day 22p.i. and dropped to the lowest at the peak phase; while M2-treated group had lost weight until day 17p.i. and then their weight increased gradually. On day 22 p.i., EAE symptoms reached a peak with average scores of 3.08±0.20. The M2-treated group showed the highest score on day 17 p.i., with an average score of 2.36±0.26(P<0.0001)(Figure 2b).
During the peak hour, the spinal cord was removed for pathological staining after perfusion. As shown in Figure 2c, the inflammatory cell infiltrations in spinal cords were less in the M2-treated group than in EAE control group measured by HE staining(P<0.0001). LFB staining showed that spinal demyelination in the M2-treated group was significantly less severe than that in the EAE control group(P<0.0001).
Additionally, pro-inflammatory cytokines, IL-6, IL-17A, TNF, IL-1ß and IL-12p70, are lower in the M2-treated group compared with the controls(P<0.01). Anti-inflammatory cytokines, IL-4 and IL-10 are higher in M2 treated group(P<0.01)(Figure 2d).