FIGURE 8.

RvD1 potently attenuates M1 inflammatory markers while increasing the functional M2 marker. A, on day 22 post- immunization, spinal cords were isolated from both groups, and the expression of pro-inflammatory M1 cytokines, M2 gene, myelin genes, and neurotrophic factors was analyzed and normalized against the L27 housekeeping gene using quantitative PCR (n = 4). B, infiltrating mononuclear cells were isolated from RvD1-treated and -untreated groups, and the cellular M2 phenotype (CD206⁺ and scavenger receptor-positive) was profiled using FACS analysis for activated myeloid cells, including monocytes/macrophages (CD11b⁺CD45^hi); C, resident microglia (CD11b⁺CD45_low) (n = 4). D, infiltrating mononuclear cells were profiled for myeloid-derived suppressor cells (CD45^hi CD11b⁺ Gr1⁺) and their M2 phenotype (CD206⁺ and scavenger receptor-positive) using FACS analysis (n = 4). E, rat mixed glial culture was treated with RvD1 (100 nm) for 2 h followed by treatment with pro-inflammatory cytokine combination (TNFα and IFNγ; 20 ng/ml). Post-24 h of incubation, RNA was isolated, and expression of inducible NOS (iNOS) and myelin genes (MOG and PLP) was normalized with L27 as a housekeeping gene (n = 4). F, panel i, primary mouse brain microglial cells were treated with vehicle (0.001% ethanol) or RvD1 (100 nm). Post-24 h of incubation, RNA was isolated, and expression of arginase 1 and chitinase-3-like-3 (YM1/2) was normalized with L27 as a housekeeping gene. F, panel ii, for the coculture study, treated and untreated microglial cells with RvD1 were washed three times with complete culture media, and primary rat oligodendrocyte progenitor cells (OPCs) were cocultured. After 24 h of incubation, cells were processed for expression of myelin genes, including myelin-associated glycoprotein (MAG), myelin oligodendrocyte glycoprotein (MOG), and myelin basic protein (MBP) by quantitative PCR, and data were normalized to the control gene ribosomal L27 housekeeping gene. ***, p < 0.001, and **, p < 0.01.