Dba 1j mice

DBA/1J mice (age, 8–12 weeks) were purchased from Charles River Laboratories (Beijing, China) and maintained in a specific, pathogen-free animal facility at Shaanxi Normal University. All animal procedures were approved by the Institutional Animal Care and Use Committee of Xi’an Jiaotong University for the use of laboratory animals. CIA was induced according to standard protocol. Briefly, mice were immunised intradermally at two sites at the base of the tail with a total of 200 μL type II chick collagen (2 mg/ml; CII; Sigma-Aldrich) emulsified in complete Freund’s adjuvant (CFA) containing 5 mg/mL heat-killed Mycobacterium tuberculosis (H37Ra; Difco Laboratories, Detroit, MI, USA). On day 21, these mice were given a boost immunization of 80 μL CII dissolved in CFA. Beginning on day 1 after the second immunization, rmIL-25 (0.5 μg/mouse) was consecutively and intraperitoneally injected for 5 days. Mice were monitored daily for signs of arthritis. Clinical arthritis was evaluated using the following scale: grade 0 = no swelling; grade 1 = slight swelling and erythema; grade 2 = pronounced swelling; and grade 3 = joint rigidity. Mice were sacrificed on day 36; knee joints were dissected and processed for RNA extraction, as well as for histologic studies, and serum was collected for ELISA.

Mice transgenic for bovine GH (bGH) under the control of the phosphoenolpyruvate carboxykinase promoter on a C57BL/6J background (18 (link)) were maintained by continuous backcrosses on C57BL/6J females. 35 transgenic mice (GHTg) and 33 control littermates (10–14 weeks old) were used, with matched sex ratios in each experiment. DBA/1J mice (50 males) were obtained from Charles River Laboratories International. Three OVA-specific TCR-transgenic mice (OT-II) were donated by Dr. C. Ardavín (Centro Nacional de Biotecnología, Madrid, Spain). Mice were handled according to national and European Union guidelines, and experiments were approved by the Comité Ético de Experimentación Animal, Centro Nacional de Biotecnología/CSIC and the Regional Government (PROEX 250-16).

Female 7-week old DBA1/J mice weighing 18–20 g were purchased from Charles River, Canada, and acclimatized for 7 days prior to the start of the experiments. Four mice per cage were housed and maintained in a temperature-controlled (24 ± 1 °C) room with light cycles of 12 h (7:00 AM–7:00 PM) and 30 to 50% humidity. Applying refinement to the use of animals in arthritis research, we provided breeder diet, food and water, soft diet on the cage floor, and soft bedding during the whole study [36 (link)].
Arthritis induction was done by intradermal injection of an emulsion consisting of bovine type II collagen in complete Freund’s adjuvant (CFA), followed 21 days later by a booster immunization with type II collagen emulsified in incomplete Freund’s adjuvant (IFA). On day 25, to synchronize onset of arthritis, 2 mg/kg lipopolysaccharides (LPS) from Escherichia coli O111:B4 (Sigma) in 100 μL saline was injected intraperitoneally to synergistically trigger and enhance arthritis [37 (link)].

DBA/1J female mice, 8–12 weeks old, were supplied by Biogem SCARL (Ariano Irpino, Italy). C57BL/6 female mice and additional female DBA/1J mice were purchased from Charles River (Wilmington, MA, United States) and housed in the animal facility of the University of Perugia. CIA was elicited by intradermal immunization with 50 μL/mouse collagen type II (Sigma, C9301-5MG, Lot#016M4158V; St. Louis, MO, United States) emulsified in complete Freund’s adjuvant (Sigma, F5881-10ML, Lot#SLBQ1106V) on day 1 and in incomplete Freund’s adjuvant (Sigma, F5506-10ML, Lot#SLBL9742V) on day 31. Arthritis was induced in 15 mice (3 groups of 5 mice each), while 3 mice composed the untreated negative control group. An additional 12 mice (4 mice/group) were immunized and utilized for gene expression profiling. Complete details of CIA mouse generation are provided in the Supplementary Material. Details of the CIA experimental procedure, with the arthritis score for each mouse, are given in Supplementary Material. Animal experiments were performed after approval by the ethical committee of the University of Perugia.

The CIA mouse model was established as previously described (Wang C. et al., 2020 (link)). Briefly, 7–8 weeks old male DBA/1J mice (Vital River Laboratory Animal Technology, Beijing, China) were intradermally injected with 100 μg of bovine type II collagen and Freund’s complete adjuvant in a 1:1 ratio (vol/vol) emulsion (100 μL) at the base of the tail. DBA/1J mice were intraperitoneally injected with 200 μg bovine type II collagen on Day 21 after the first immunization. In the therapeutic treatment study, the mice were randomly divided into groups. Myr group (100 mg × kg⁻¹, every day, n = 5) or DMSO group (vehicle, n = 5) was intragastrically administered for 14 days. Arthritis progression was monitored every other day according to previously described scoring system ranging from 0 to 4 (0, normal; 1, swelling or redness of paw or a single digit; 2, 2 joints involved; 3, 3 joints involved; 4, severe arthritis of the entire paw and digits) (Thornton et al., 2017 (link)). The arthritic score was independently calculated in a blinded manner and defined as the combining scores of all four paws. All mice were raised under specific pathogen-free (SPF) conditions. All experiments were performed in accordance with institutional guidelines and approved by the Institutional Animal Care and Use Committee of the First Affiliated Hospital, Sun Yat-sen University (SYSU-IACUC-2020-000153).

Six- to 12-week-old healthy mice (C57BL/6J background, specific pathogen-free (SPF)) were purchased from the Experimental Animal Center of Sun Yat-sen University; DBA/1J mice (4–5 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). Mice were kept in individually ventilated cages in an SPF environment with a 12 h light–dark cycle at a constant temperature of 25 °C. The mice were acclimated for 7 days in this new environment prior to the formal experiment. The cages instead of animals are randomly divided into different groups because mice from different cages may fight with each other. The severity of the diseases was recorded by investigators who were blinded to the group allocations. Investigators were not blinded to the ex vivo experiments of cells from the animals. The experimental procedures employed in this study were approved by the Ethical Committee of Sun Yat-sen University (Approved Nos. 2019-3138, 2019-683, and 2020-0698).