Mycobacterium butyricum

All experimental procedures were approved by the Animal Care and Use Committee of the Université de Sherbrooke and were in accordance with the policies and directives of the Canadian Council on Animal Care and guidelines from the International Association for the Study of Pain (IASP). Adult male Sprague–Dawley rats (200–225 g, Charles River) were housed two per cage in a climate‐controlled room on a 12 h light/dark cycle with free access to food and tap water. Complete Freund's adjuvant (CFA, Calbiochem) was prepared with 7 mg/ml of Mycobacterium butyricum (Difco Laboratories) and emulsified 1:1 with saline 0.9%. Rats under light anesthesia with isoflurane received an intraplantar injection of 100 μl (400 μg) of the freshly emulsified CFA mixture into the left hind paw. Sham animals received an intraplantar injection of 100 μl of saline.

Adjuvant arthritis (AA) was induced to male Lewis rats weighing 160–180 g by a single intradermal injection of 0.1 mL suspension of heat-inactivated Mycobacterium butyricum (Difco Laboratories, Detroit, MI, USA) in incomplete Freund’s adjuvant at the base of the tail.

Complete Freund’s adjuvant (CFA) (Calbiochem, La Jolla, CA, USA) was complemented with 7 mg/mL of mycobacterium butyricum (Difco laboratories, Detroit, MI, USA), then emulsified 1:1 with saline 0.9%. Under light anesthesia (isoflurane 5%), rats received an intraplantar injection of 100 μl of the freshly emulsified mixture. Pain-related behaviors were assessed on the same animals before and on days 3, 8, 9, and 10 following CFA injection. Animals received an i.t. injection of either vehicle or 45 μg/kg INCB3344 on days 8, 9, and 10 1 h before behavioral examination. Sham animals received an intraplantar injection of 100 μl saline.

Monoarthritis was induced as previously described [9 (link),56 (link)]. Under brief anesthesia with isoflurane (5% for induction and 3% for maintenance; Isoflo, Abbott Animal Health, Madrid, Spain), rats (n = 50) were injected in the left tibiotarsal joint with 50 μL of CFA solution (30 mg of desiccated Mycobacterium butyricum from Difco Laboratories, Detroit, MI, USA, diluted in the vehicle solution containing 3 mL paraffin oil, 2 mL saline, and 500 μL Tween-80). The control group (n = 51) was injected with CFA vehicle. Throughout the experimental period, the animals that showed signs of polyarthritis were immediately removed from the study.

This study was performed in accordance to the legislation of the European Community and was approved by the Ethical Committee for Animal Experiments of Saint-Etienne University (registered to the French government as #0125.01). All methods were performed in accordance with these guidelines and regulations. Six-week-old female Lewis rats (Janvier Laboratories, Saint-Germain-sur-l’Arbresle, France) were housed in the PLEXAN facility with 12/12 h light/dark cycles and ad libitum water and food access. After 7 days of housing and acclimation training, rats were randomly separated into two groups. In the rat AIA (n = 35), arthritis was induced by subcutaneous injection of 300 µL of 5 mg.mL⁻¹Mycobacterium butyricum (Difco Laboratories, Detroit, MI, US) in mineral oil. Control rats (CTRL group, n = 35) received only mineral oil. Injection was performed under short and light 2% isoflurane anaesthesia to optimize the full deliverance of the dose and obtain a high rate of arthritis induction^{12 (link)}. The day of arthritis induction was defined as day 0. Seven time points were defined: 0, 6, 8, 10, 12, 17, and 24 days. Five AIA and 5 CTRL rats were sacrificed at each time point to perform histological and biochemical analysis.

The adjuvant-induced arthritic (AIA) rat model in this study was based on that described by Sano et al. and Turull and Queralt [25 (link),60 (link)]. In this study, 10 mg/mL heat-killed and lyophilized Mycobacterium butyricum (DIFCO Laboratories, Detroit, MI, USA) was suspended in incomplete Freund’s adjuvant (Sigma-Aldrich) maintained on ice. Rats were then immunized by adjuvant injection of 10 mg/mL M. butyricum in incomplete Freund’s adjuvant. On Day 0, rats were injected intra-dermally at the base of the tail with 0.1 mL of adjuvant, and the development of arthritis was monitored from Day 0 to Day 28. The rats were randomly divided into 5 groups: AIA group (n = 7), AIA + Exc-B group (2.5 mg/kg) (n = 6), AIA + Exc-B group (5 mg/kg) (n = 6), control group (n = 6), and Exc-B (5 mg/kg) group (n = 6).