Chicken type 2 collagen

Chicken collagen type II (Chondrex), Freund's complete adjuvant (Sigma‐Aldrich), Freund's incomplete adjuvant (Sigma‐Aldrich), FBS (Gibco), RPMI 1640 (Gibco), erythrocyte lysate (Beyotime), Masson staining solution (NanJing JianCheng Bioengineering Institute), PrimeScript™ RT Maser Mix, SYBR Premix Ex Taq™ (TaKaRa), TRIzol Reagent (Invitrogen), Pierce™ BCA Protein Assay Kit (Thermo Scientific™), anti‐MEKK2, Foxp3, ROR‐γt, IL‐17A and GAPDH (Abcam), blocking buffer of SuperBlock™ T20 (PBS), Blocking Buffer (Thermo Scientific), prestained protein Marker (Thermo), secondary antibody (HRP‐Goat‐Rabbit Jackson ImmunoResearch), antimouse CD3‐FITC, antimouse CD4‐PerCP‐cy5.5 and antimouse IL‐17‐A‐APC (eBioscience), and Cell stimulation Cocktail 500× (eBioscience).

Herein, 8-week-old DBA/1 mice were obtained from the Beijing Huafukang Biotechnology Company (Beijing, China). Thirty male mice were randomly divided into two groups: CIA group (n = 15) and normal group (n = 15). The CIA model group underwent the following induction [42 (link)]. An intraperitoneal injection of anesthesia and pentobarbital sodium was conducted, after which, chicken-type II collagen (Chondrex, USA) was emulsified to complete Freund’s adjuvant (Chondrex, USA) at a ratio of 1:1, and 100 µL of emulsifier was injected subcutaneously at the root of the tail. After 21 days, an emulsifier of chicken type II collagen and incomplete Freund’s adjuvant were added (Chondrex, USA). Scores were assessed according to the following standards [43 (link)]. When the CIA mice developed joint swelling (score ≥ 4), the CIA and normal mice were euthanized to isolate BMDMs. All animal experiments were approved by the Animal Ethics Committee of West China Hospital, Sichuan University (Nos. 2,020,304 A).

We established our mouse model of periodontitis with CIA following previous protocols.19 The bacterial strain used was Pg (ATCC: 33277), which was grown in an anaerobic chamber (80% N₂, 10% H₂ and 10% CO₂) at 37°C. Before the formal oral bacterial colonization, kanamycin (0.5 mg/mL) was added to drinking water for three consecutive days in order to remove other bacteria. The bacterial pellet obtained by centrifugation was mixed with an equal volume of sterile 3% carboxymethylcellulose (CMC) and topically applied in the oral cavity and anus eight consecutive times. The dose of the mixture was 100 μL (5 × 10¹⁰ cells/mL of Pg) per mouse.23 Following bacterial application, chicken type II collagen (Cat#20011, Chondrex) was emulsified in 100 μg complete Freund's adjuvant (Cat#7023, Chondrex). Fifty microlitres of the emulsion was slowly injected intradermally at a point about 1.5 cm distal from the base of the tail. The primary immunization was performed one day after bacterial application, and the booster immunization was made after 14 days.24

Samples prepared with Laemmli buffer were heated 2 h at 50 °C and resolved by SDS-PAGE using Mini-PROTEAN^® TGX™ Precast Protein 4–15% gradients gels (Bio-Rad). Chicken type II collagen (Chondrex) was used as a reference to validate its immunodetection from media taken from tissue culture supernatants. Proteins were transferred to polyvinyl (PVDF) membranes using the Trans-Blot Turbo western blotting transfer system (Bio-Rad). PVDF membranes were stained with Revert Total Protein (Licor) and blocked for 1 h in 1X Tris-buffered saline (TBS) with 5% non-fat milk (Bio-Rad) at room temperature. PVDF membranes were incubated overnight at 4 °C with a mouse monoclonal anti-type II collagen antibody (10 µg/ml, #7005, Chondrex) according to the manufacturer’s indications. Three times washes were done for 10 min each in 1X TBS with 0.1% Tween-20 previous detection using the corresponding peroxidase-conjugated anti-mouse antibody for 1 h at room temperature. Type II collagen was detected with the ECL supra bright kit (Agrisera). Chemiluminescent signal on PVDF membranes was detected using the Odyssey Fc Imaging system (Licor), and the intensities of the bands after removal of background intensities were quantified using ImageStudioLite software (Licor) and normalized to the total amount of protein in each lane.

Chicken type II collagen and complete Freund adjuvant were purchased from Chondrex. H&E staining kit was from Jiancheng Bio. Safranine red and Fast Green staining kit was from Solarbio. Dulbecco’s modified Eagle’s medium (DMEM) was from Gibco. Fetal bovine serum (FBS) was from Wisent. Diaminobenzidine peroxidase substrate kit was from Vector Laboratories. Transfection reagent GenJet was from SignaGen. Bicinchoninic acid protein assay kit was from Thermo Fisher. Enhanced chemiluminescence detection kit was from Vazyme. Cytokines such as TNF-α, IL-1β, IL-6, and LPS were from Peprotech. Nuclear and cytoplasmic extraction reagents were from Thermo Fisher. The antibody against DEC1 was described elsewhere (61 (link)). Anti-CTSK was from Santa Cruz Biotechnology. Anti-NFATc1 was from Cell Signaling Technology. All other antibodies were from BioGot. Human IL-6 ELISA Kit was from SHRBIO.

Mice were immunized with 100 μg (2 mg/ml) chicken type II collagen (Chondrex, Redmond, WA, USA) emulsified in an equal volume of complete Freund’s adjuvant (CFA) containing 1 mg/ml heat killed Mycobacterium tuberculosis (strain H37Ra; Difco), administered intradermally (i.d.) in the tail base on day 0 and subcutaneously (s.c.) on the mouse back on day 21. Mice were killed on day 35. Mice were considered to have arthritis when significant changes in redness and/or swelling were noted in the digits or in other parts of the paws. Arthritis was scored visually using the following scale: 0, non-inflamed; 1, mild inflammation; 1.5, marked inflammation; 2, severe inflammation. Arthritis development was scored macroscopically, with a maximum possible score of 8 per mouse. Mice with a score of ≥ 6 were killed for ethical reasons. Scoring was performed blindly by two independent observers, without knowledge of the experimental groups [18 (link)].