Heat killed mycobacterium tuberculosis

EAE was induced as described previously [1 (link)]. Briefly, mice were injected subcutaneously with 250 μg MOG35-55 peptide (Lysine Bio-system, Xian, China) emulsified in complete Freund's adjuvant (CFA, Sigma, St. Louis, USA) containing 4 mg/ml of heat-killed Mycobacterium tuberculosis (Difco Laboratories, Detroit, MI, USA). At 0 hour and 48 hours after immunization, mice were injected intraperitoneally with 500 ng pertussis toxin (Alexis, San Diego, CA, USA).
Randomized treated intraperitoneally once per day with vehicle or drugs - SB203580 at 5 mg/kg body weight, TEMPOL at 25 mg/kg body weight. The mice were examined daily for clinical signs of EAE and scored as follows: the scale ranged from 0 to 15 and is the sum of the state of the tail and all of the four limbs. For the tail the following scoring was followed - 0 reflected no signs, 1 reflected a half paralyzed tail, and score of 2 reflected fully paralyzed tail. For each of the hind-or forelimbs, each assessed separately, 0 signified no signs, score 1 signified weak or altered gait, score 2 signified paresis, while a score of 3 signified fully paralyzed limb. A fully paralyzed quadriplegic animal would thus a score of 14. Mortality equals a score of 15.

For induction of CIA, we used the immunization protocol for C57BL/6 strain (H-2b)57 (link). Briefly, mice aged between 8 and 10 weeks of age were injected intradermally at several sites into the base of the tail and back with type II collagen (Sigma-Aldrich, Cat. no. C9301) emulsified in complete Freund adjuvant: incomplete Freund adjuvant (GIBCO), heat-killed mycobacterium tuberculosis (Difco Laboratories) on day −21 and the same injection was repeated on day 0 (Fig. 1a and Supplementary Fig. 2). Arthritis development in each paw was scored by macroscopic evaluation58 as follows: (0) no change, (1) erythema and mild swelling confined to the ankle, (2) erythema and mild swelling from the ankle to midfoot, (3) moderate swelling and (4) severe swelling. The maximum score per mouse is 16. The investigators (M.M., K.S., S.N. and J.S.H.) were blinded to the genotypes. Ten to twenty mice/genotype were used (Fig. 1 legend and Supplementary Fig. 2 legend). Mice were dissected 2 weeks after the second immunization to evaluate the draining lymph nodes (popliteal, inguinal, axillary and brachial).

Twelve 7- to 8-week-old male DBA/1J mice from Jackson Laboratories (USA) were used. The institutional IACUC committee approved all the procedures carried out. First, incomplete Freund’s adjuvant (IFA), which is a mixture of 15% Arlacel A and 85% mineral oil, was prepared (Sigma); then, heat-killed Mycobacterium tuberculosis (BD Bioscience, CA, USA) was added to IFA at a final concentration of 4 mg/ml to make complete Freund’s adjuvant (CFA). CFA and type ІІ collagen (Chondrex, WA, USA) were emulsified at a 1:1 ratio by a tissue homogenizer to make the final emulsion for injection. Fifty microliters of this emulsion was injected intradermally (i.d.) into the tail of each mouse at approximately 1.5 cm distal to the base of the tail [17 (link)]. The thickness of each affected hind paw was measured with microcalipers [18 (link)]. The paws of each mouse were clinically scored with the following scale of 0–4: 0 = normal, 1 = slight erythema and edema, 2 = increased edema with the loss of landmarks, 3 = marked edema, and 4 = marked edema with ankylosis of the joint. Finally, the articular index, which is the sum of the scores for all four paws of each mouse, was determined [19 (link)].