Caspase 1

BMDMs were isolated from bone marrow of C57BL/6 wild-type or Caspase-1^−/− (Jackson Laboratory) female mice (7–8 weeks old) and differentiated for 7 days in RPMI 1640 medium supplemented with 10% fetal bovine serum (Gibco), penicillin (100 U/ml) and streptomycin (0.1 mg/ml) and 20 ng/ml human M-CSF (R&D Systems). THP-1 (Cell Bank of Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences) were cultured in RPMI 1640 medium with FBS and penicillin and streptomycin. Phorbol 12-myristate 13-acetate (100 ng/ml, 24 h) was used in differentiation of THP-1 into macrophages. The additives or inhibitors used in in vitro experiments are as follows: purified SAK (0.5 μg/ml), lipopolysaccharide (LPS, 0.2 μg/ml), ATP (5 mM), MCC950 (1 μM, Selleck), potassium channel blocker glibenclamide^{49 (link)} (10 μM, Sigma), KCl (25 mM), NADPH oxidase inhibitor apocynin^{50 (link)} (200 μM, Selleck), lysosome membrane stabilizer dexamethasone^{51 (link)} (200 nM, Sangon Biotech), ROS scavenger NAC^{52 (link)} (1 mM, Sigma), and NF-κB inhibitor BAY 11-7082 (10 μM, Sigma).

The p47^phox-deficient (p47^phox-/-, #004742), NLRP3^-/- (#017970), Caspase-1^-/- (#016621), IL-18^-/- (#004130), IL-1R1^-/- (#003245) and wild type (WT) mice were on C57BL/6 background and were purchased from the Jackson Laboratory (Bar Harbor, Maine, USA). Mice were maintained in micro-isolator cages, fed with standard laboratory chow diet and water, and housed in the animal colony at the animal center of the Third Military Medical University (TMMU). Mice approximately 12 weeks of age were used for these experiments. All animals received humane care according to the criteria outlined in the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86–23 revised 1985).

C57BL/6, CD45.1, Ifnar1^-/-, Stat2^-/-, Il10^-/-, Il10rb^-/-, Il1r1^-/-, Caspase-1^-/-, Ccr2^-/-, Csf2^-/-, Il27ra^-/-, GFP⁺, and Rag1^-/- mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Stat1^-/- mice were purchased from Taconic Farms (Hudson, NY, USA). To generate LysM-Cre⁺Ifnar1^fl/fl mice, LysM-Cre mice were crossed with Ifnar1^fl/fl mice and used for EAE experiments. Mice were kept in specific pathogen–free conditions in 12/12 h of light/dark cycles with food and water ad libitum. All experimental breeding and procedures were performed with the approval of the Institutional Animal Care and Use Committee of Thomas Jefferson University.
Mice were immunized for EAE induction by subcutaneous injection of 200 µg MOG_35-55 (Genscript, CA, USA) emulsified in CFA. Mice received 200 ng of pertussis toxin (Sigma-Aldrich, MO, USA) on days 0 and 2 p.i. and were scored and weighed daily. Mice were scored according to the following scale: 0, no sign of clinical disease; 1, paresis of the tail; 2, paresis of one hind limb; 3, paresis of both hind limbs; 4, paresis of the abdomen; 5, moribund/death.