Chicken type 2 collagen

Chicken collagen type II (4 mg ml⁻¹; Sigma) was emulsified in equal amounts with complete Freunds adjuvant (Sigma) containing 5 mg ml⁻¹ heat-inactivated Mycobacterium tuberculosis (H37Ra; Difco). For the induction of CIA, male 7-week-old DBA/1J mice (Janvier) were injected s.c. at the base of the tail with 100 μl of this emulsion. Mice were rechallenged after 21 days. N-acetylmannosamine was constantly administered to the drinking water at a concentration of 10 g l⁻¹, beginning with the primary immunization. Control mice received water or water containing 10 g l⁻¹ mannose (all Sigma). The allocation of the mice into the different groups was performed randomly. Clinical arthritis was evaluated every second day using a scoring system from 0 (no swelling) to 3 (severe swelling and erythema) for every limb, resulting in a maximally possible score of 12 per animal.

Type II chicken collagen (Sigma, USA) was dissolved in 0.1 M acetic acid at 4 mg/mL overnight at 4°C under sterile condition. Each rat was immunized by intradermally injection of 100 μg CII emulsified in CFA (Sigma, USA) at the base of the tail on day 0. A booster injection was conducted 7 days after the primary immunization with another 100 μg CII emulsified in IFA (Sigma, USA). To evaluate the influence of rhIL23R-CHR on the treatment of CIA model, rats were treated with intravenous administration of rhIL23R-CHR at 1 mg/kg every two days from day 0 to day 20. CsA was given by intragastric administration at 1.5 mg/kg as a positive control. Normal and CIA rats were administrated with an equal volume of PBS at the same time. The severity of the arthritis was scored every two days. Inflammation of two hind paws were graded from 0 to 4: grade 0, paws with no swelling and focal redness; grade 1, paws with swelling of finger joints; grade 2, paws with mild swelling of ankle or wrist joints; grade 3, paws with severe inflammation of the entire paws; and grade 4, paws with deformity or ankylosis. Each paw was graded and the two scores were combined so that the maximum possible score per rat was 8. Clinical scores were based on the sum of two hind paws. In addition, body weight and the diameter of ankle thickness were recorded.

To evoke collagen-induced arthritis (CIA) in BALB/c mice, the procedure described by Takahashi et al. [17 (link)] was employed. Briefly, mice (7 per group) were immunized six times subcutaneously into the inguinal region with the mixture containing 200 μg of type II chicken collagen (Sigma-Aldrich, USA) and 100 μg of tested LPS in a total volume of 150 μl, at the intervals of 30 days. Mice immunized with CII only or injected with PBS with no antigen were used as controls. Twenty days after last immunization, mice were weighted, tested in CatWalk gait analysis system (see below), and sacrificed. Blood for serum isolation, lymph nodes, and livers were isolated. All possible steps were taken to avoid animal suffering at each stage of the experiment.

Type II chicken collagen (Sigma) at 4 mg/mL was emulsified 1:1 with Complete Freund's Adjuvant (Sigma). C57BL/6 mice were injected ip at the base of the tail with two injections of the emulsion at 50 μL per site. The mice were observed daily for signs of ill health and were scored for clinical signs of arthritis three times a week after day 18. The severity of arthritis was graded using an established scoring system³⁵: 0 = no arthritis, 1 = 1 inflamed digit, 2 = 2 inflamed digits and/or erythema and mild swelling of the footpad, 3 = >2 digits and footpad inflamed, 4 = all digits and footpad inflamed. An arthritis score for each mouse was calculated by summing up the scores for each paw. If the score was 12 or higher, the mouse was immediately euthanized in accordance to the Home Office guidelines.

The induction of collagen induced arthritis (CIA) in the C57BL/6 strain was performed as previously published [17 ]. Male WT and Ptpn22^−/− mice of 10–14 weeks of age were injected intradermally at the base of the tail with 100 μg chicken type II collagen (Sigma) emulsified in complete Freund's adjuvant. Clinical signs of arthritis were assessed visually in the wrist and ankle joints 3 times weekly, using a previously described severity scale: 0 = no arthritis; 1 = 1 inflamed digit; 2 = 2 inflamed digits; 3 = more than 2 digits and footpad inflamed; 4 = all digits and footpad inflamed [17 ]. Scoring was conducted under blinded conditions for up to 96 days. At day 96 single cell suspensions from lymph nodes (LN) and spleens were restimulated for 6 h with PMA (Sigma; 50 ng/ml) ionomycin (Sigma; 10 ng/ml) and monensin (Biolegend; 1 in 1000) and expression of IFNγ (clone XMG1.2; Biolegend), IL-17 (clone TC11-18H10.1; Biolegend), TNFα (clone MP6-XT22; Biolegend), IL-4 (clone 11B11; Biolegend) and IL-10 (clone JES5-16E3; Biolegend) determined by intracellular flow cytometry.

The male DBA/1 mice were immunized by an intradermal injection at the base of the tail with 100 μg chicken type II collagen (Sigma‐Aldrich) emulsified in Freund's complete adjuvant (Sigma‐Aldrich) as previously described 4. On day 21 after the first immunization, the mice were boosted in the same way using the collagen emulsified in Freund's incomplete adjuvant. When the mice showed visible clinical signs resulting in a clinical score of ≥3, treatment was started. Resveratrol and tetrandrine were equably suspended in 0.5% carboxymethylcellulose sodium (CMC‐Na). To investigate the role of AhR in the tetrandrine‐mediated regulation of the activities of STAT3 and STAT5 in the CIA mice, the resveratrol was orally administered at 20 mg/kg, and the tetrandrine was orally administered at 40 mg/kg per day for 14 consecutive days from the day 28 after the first immunization. The vehicle group was orally administered an equivalent volume of 0.5% CMC‐Na.