Fig 2: CSF1R antagonism differentially affects gene expression in CNS compartments. A-D Cerebella and spinal cords were isolated from CD and PD mice at onset and acute EAE and RNA transcripts were analyzed by quantitative PCR. Data are shown as means ± SD, n = 6–12; *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.00005

Fig 3: CSF1R antagonism increases CNS infiltration by myeloid cells before neurological symptoms appear in EAE. Single cells isolated from pooled brain and spinal cord tissues were analyzed by flow cytometry for myeloid subsets before onset of neurological deficits (preonset) A Numbers of total cells, and of infiltrating immune cells (CD45hi), which include myeloid cells (CD45hiCD11b+) and lymphocytes (CD45hiCD11b-) were increased while microglia were decreased in the CNS of PD compared to CD mice in preclinical EAE. B Gating strategy for flow cytometric analysis of myeloid cells in the CNS. C Frequencies and absolute numbers of neutrophils (CD45hiCD11b+Ly6G+) were increased in the CNS of PD mice with during preonset EAE compared with CD mice. D No differences in the frequencies of total monocytes (CD45hiCD11b+Ly6G-CD11c-MHCII-), total macrophages (CD45hiCD11b+Ly6G-CD11c-MHCII+) and total DCs (CD45hiCD11b+Ly6G-CD11c+MHCII+) were detected but their numbers were increased in the CNS of PD compared to CD. E Frequencies and numbers of cDCs (CD45hiCD11b+Ly6G-CD11c+MHCII+CD26+CD88-), but not of moDCs (CD45hiCD11b+Ly6G-CD11c+MHCII+CD26+CD88+) were increased in the CNS of PD compared to CD mice. F Numbers of Ly6C+ monocytes, and of both Ly6C+ and Ly6C- macrophages were increased in the CNS of PD compared to CD mice in preclinical EAE. Representative flow plots are shown. Both male and female mice were employed. Data are shown as means ± SD, n = 22; *p < 0.05, ***p < 0.0005, ****p < 0.00005

Fig 4: Effect of CSF1R antagonism on the expression of CSF1R and its ligands during EAE. A Csf1r transcript levels are lower in the PD compared to CD CNS during EAE. There were no differences in the transcripts of Csf1 and Il34. B CSF1R (CD115) expression was detected using flow cytometry in Ly6C+ and Ly6C- microglia, monocytes, macrophages, and neutrophils in pooled brain and spinal cord (CNS), and SLOs during EAE. C Quantification of B. Data are shown as means ± SD, A: n = 9–12; B: n = 16 *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.00005

Fig 5: CSF1R antagonism ameliorates EAE clinical severity. A Schema of experimental design. B Classical EAE neurological symptoms onset was delayed, and severity was milder in PD compared to CD mice. C Peak and cumulative EAE scores were lower in PD compared to CD mice. D Microglia (CD45loCD11b+) are depleted in pooled brain and spinal cord tissues of PD mice in preclinical and acute EAE. E Analysis of Ly6C expression in CD45loCD11b+ cells shows that CD45loCD11b + Ly6C- cells were preferentially depleted in the CNS of PD mice. Representative flow plots are shown. Both male and female mice were employed. Results are shown as means ± SD; n = 44–47 CD, 40–49 PD; *p < 0.05, ****p < 0.0005