The 13-week-old female mice were immunized subcutaneously with 150 μg of MOG35~55 peptides (Cambridge BioSciences, Cambridge, UK) emulsified in CFA containing heat-killed MTB (0.4 mg per mouse, Chondrex, Redmond, WA, USA). Mice were injected intraperitoneally with 500 ng of pertussis toxin (Kaketsuken, Kumamoto, Japan) on days 0 and 2 (39). Mice were observed daily for clinical signs of disease and euthanized at the peak of disease. Disease severity was recorded as follows: 0, normal; 1, limp tail; 2, wobbly gait; 3, hind limb weakness; and 4, hind limb paralysis. In IL-1Rα treatment experiments, Vsig4−/− mice were subcutaneously injected with IL-1Rα (#200-01RA, 200 ng per mouse per week, PeproTech) or saline solution (vehicle) starting 1 day before MOG35~55 peptide administration. In VG11 mAb therapeutic experiments, C57BL/6 WT female mice were induced to develop EAE by MOG35~55 peptides; mice also received VG11 mAbs (100 μg per mouse per week) or mouse isotype IgG1 antibodies (100 μg per mouse per week) by intraperitoneal injection. At day 30, these mice were euthanized and serum was collected. PEMs were also isolated from these EAE mice and seeded into six-well plates at a density of 2 × 105 cells per well. Cell culture supernatants were collected to detect the secretion of IL-1β after 12 hours. For histopathological analyses, the spinal cords were fixed in 4% formaldehyde, decalcified in EDTA, embedded in paraffin, sectioned, and stained with H&E. In some experiments, mononuclear cells were isolated from the spinal cords and spleen tissues of EAE mice following perfusion with PBS by Percoll density centrifugation, and cell infiltration was detected by flow cytometry.

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