Mog35 55 peptide

Active EAE was induced in 12-week-old female mice by s.c. immunization at two sites, upper and lower back, with 200 μg MOG_35–55 peptide emulsified in CFA containing 400 μg heat-killed Mycobacterium tuberculosis H37Ra (Hooke Laboratories, Lawrence, MA, USA), on day 0. In addition, mice received 200 ng pertussis toxin (Hooke Laboratories) i.p. in 0.1 ml per mouse on days 0 and 1. Clinical signs of EAE were assessed daily with a 0- to 5-point scoring system defined as follows: 0, no obvious changes in motor function compared with non-immunized mice; 0.5, tip of tail is limp; 1, limp tail; 1.5, limp tail and hind leg inhibition; 2, limp tail and weakness of hind legs; 2.5, limp tail and dragging of hind legs; 3, limp tail and complete paralysis of hind legs or paralysis of one front and one hind leg; 3.5, limp tail and complete paralysis of hind legs that are together on one side of body; 4, limp tail, complete hind leg and partial front leg paralysis; mouse is still minimally moving and appears feeding; 4.5, complete hind leg and partial front leg paralysis; no movement around the cage, mouse is not alert; 5, death or severe paralysis. Mice with score ≥4 for two consecutive days and mice with score 5 were killed.

The Animal Policy and Welfare Committee of Chengdu Medical College approved all animal experiments (Approval ID: 2,021,711). Seven-week-old female C57/BL6 mice (weight, 18–21 g) (GemPharmatech Co., Ltd., Chengdu, China) and Nlrp3^−/− mice on a C57BL/6 (Weishanglide Biotechnology Co., Ltd., Beijing, China) were raised at the Animal Experimental Center of Chengdu Medical College. We established the EAE model as described (Yu et al., 2020 (link)). Briefly, 100 μl of Complete Freund’s Adjuvant (CFA; Chondrex, Woodinville, WA, United States) was ultrasonically emulsified with 400 μg of Mycobacterium tuberculosis H37Ra (BD Biosciences, San Jose, CA, United States). Thereafter, 200 μg of myelin oligodendrocyte glycoprotein 35–55 (MOG35-55) peptide (Hooke Laboratories, Lawrence, MA, United States) mixed with 100 μl of PBS and the emulsion, was subcutaneously injected into the mice (immunization day 1). On that day and on post-immunization day 3, 300 ng of pertussis toxin (PTX; List Biological Laboratories Inc., Campbell, CA, United States) was injected i.p. (Figure 1A).