M3761

Naive NOD scid gamma (NSG) mice were either treated with antibiotics: ampicillin (1 g/L, A9393, Sigma-Aldrich), neomycin (1 g/L, N1876, Sigma-Aldrich), metronidazole (1 g/L, M3761, Sigma-Aldrich), and vancomycin (0.5 g/L, V2002, Sigma-Aldrich); or kept on autoclaved water for three weeks. Splenocytes were isolated from arthritic K/BxN mice or NOD controls, pooled, and transferred i.v. (60 million cells/mouse) into control, antibiotics-treated mice or NSG mice reconstituted with gut microbiota from K/BxN or KRN mice as described below.
For bacterial reconstitution, NSG mice were treated with antibiotics for two weeks and changed onto autoclaved water for 24 h before bacterial reconstitution. Feces from arthritic K/BxN or control (naive KRN) mice were collected fresh, homogenized, diluted in PBS and gavaged to NSG mice (0.06 mg feces/mouse) 7 days prior and on the day of cell transfer.

AIA was induced as previously described⁴¹ (link)^,42 (link). Briefly, mice were immunized subcutaneously at the base of the tail with 200 μg methylated bovine serum albumin (mBSA, Sigma-Aldrich) in 100 μL complete Freund’s adjuvant (CFA) and phosphate-buffered saline (PBS, Sigma-Aldrich). 3 mg/ml CFA was prepared by mixing Mycobacterium tuberculosis (231141, BD) in incomplete Freund’s adjuvant (F5506, Sigma-Aldrich). After 7 days, mice received an intra-articular injection of 200 μg mBSA in 10 μL PBS in the right knee or PBS alone as a control in the left knee. Joint size was measured using calipers. Knee swelling was calculated as percentage increase in size between antigen-injected knee and PBS-injected knee.
Disease scores were calculated as follows: $\%\mathrm{S}\mathrm{w}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{i}\mathrm{n}\mathrm{g}=\frac{mBSAknee-PBSknee}{PBSknee}\times 100$ If treated with antibiotics for microbiota depletion during arthritis, C57BL/6 mice were given, in their drinking water, a combination of neomycin (1 g/L, N1876, Sigma-Aldrich), metronidazole (1 g/L, M3761, Sigma-Aldrich), and vancomycin (0.5 g/L, V2002, Sigma-Aldrich), from 7 days prior to the subcutaneous injection. The mice were kept on antibiotics throughout the experiment.

An antibiotic cocktail containing neomycin 0.5 g/L (N6386, Sigma-Aldrich, Saint Louis, MO, USA), ampicillin 0.5 g/L (A5354, Sigma-Aldrich, Saint Louis, MO, USA), metronidazole 0.5 g/L (M3761, Sigma-Aldrich, Saint Louis, MO, USA), vancomycin 0.5 g/L (SBR00001, Sigma-Aldrich, Saint Louis, MO, USA), and kanamycin 0.5 g/L (Beyotine, Shanghai, China) was prepared and applied in drinking water.

Embryos from the cross of Et(HG7L) and Tg(−8.0cldnb:NTR-hKikGR) were collected and screened for double-positive ones. We freshly prepared a 10X stock of metronidazole (Mtz, M3761, SIGMA-ALDRICH) solution (20 mM Mtz, 1% DMSO). Larvae at three days post-fertilization (dpf) were first treated with a 1X working concentration of Mtz solution (2 mM Mtz, 0.1% DMSO diluted in 0.3% PTU containing 0.3X Danieau’s buffer) for 3, 6, 9, and 12 h in the dark. Then, the Mtz solution was replaced with several washes of fresh 0.3X Danieau’s buffer for recovery. The treatment of Mtz solution was 12 h to obtain the optimal ablation result.