M tuberculosis

Female Lewis rats (6–8 weeks; ARC, Perth, Australia) were used in this study based on established protocols (Whiteley and Dalrymple, 2001 (link)), as they are the most susceptible strain to the induction of arthritis with heat-killed Mycobacterium tuberculosis. In addition, female rats tend to develop arthritis more readily than male rats, which is similar to what occurs in human rheumatoid arthritis. Animals were housed in standard laboratory cages under control conditions (12-h light-dark cycles, 22°C, 60% humidity) in groups of 4–6, with free access to food and water on tissue and shredded paper bedding. Rats were given a minimum of 7 days to acclimatize to the housing conditions. After this period rats were anesthetized via brief exposure to 2% isoflurane (Abbot, Cronulla, Australia) before receiving a single subcutaneous injection of Complete Freund's Adjuvant (CFA), containing 1 mg heat-killed M. tuberculosis (20 mg/ml) (Chondrex, Washington, USA) into the base of the tail. The experiments were approved by The University of Newcastle Animal Care and Ethics Committee.

Bovine type II collagen (CII) was bought from Chondrex and emulsified with an equal volume of complete Freund's adjuvant (CFA) containing 6 mg/ml heat-denatured M.tuberculosis (Chondrex, LLC, Seattle, WA). DBA1/J mice or DBA1/J Foxp3^gfp reporter mice were immunized via intradermal injection at the base of the tail with 100 μl of emulsion (CII 100 μg/mouse). To determine intervention effects, mice were intraperitoneally treated with 0.5 μg/mice VD (Catalog NO.71820, Cayman Chemical) dissolved in 0.1% ethanol every other day starting 1 day before immunization for 15 days or just the same volume of 0.1% ethanol as control. This dose was selected on the basis of previous experience with mouse experiments, which was found to be effective on established CIA without producing hypercalcemia (15 (link)).

All animal experiments were performed conform national guidelines and the study protocol was approved by the Ethical Committee for Animal Experimentation (Dier Experimentele Commissie; DEC) of Leiden University. DBA/1J mice were obtained from our own breeding colony (originally obtained from Charles River). C57Bl/6 mice were purchased from Charles River. CIA was induced in 8–10 week old mice by injection at the tail base with 100 µg of CII emulsified in complete Freunds adjuvant (CFA). On day 21 the mice received a subcutaneous boost with 100 µg of CII in incomplete Freunds adjuvant (IFA; Sigma-Aldrich). For DBA/1J mice bovine CII (Chondrex) and CFA containing 0.5 mg/ml of M.tuberculosis (Difco) were used and for C57Bl/6 mice chicken CII (Chondrex) and CFA containing 2.5 mg/ml of M.tuberculosis (Chondrex) were used. For the CFA only immunizations PBS was used instead of CII. Arthritis severity was monitored as described earlier using a clinical score with a maximum of 15 per paw [17] (link). For the kinetics experiments, mice were bled before immunization, on day 7, 14, 21, 28, 35, 49 after immunization and at the end of follow up. Blood was centrifuged and serum was harvested and stored at −80°C until use.