V2002

Binding of bacterial PG peptide to vancomycin or DLF-1 was monitored using an ITC₂₀₀ (Microcal, Piscataway, NJ). Vancomycin hydrochloride from Streptomyces orientalis (32 μM final concentration, V2002, Sigma) or DLF-1 (7 μM final) was diluted in 1X PBS pH 7.4 (21-040-CM, Corning), and 204 μL was loaded into the cell. Experiments contained 1% DMSO (BP-231, Fisher) in both syringe and cell, to aid solubility. Peptide Ac-L-Lys-D-Ala-D-Ala was dissolved in the same buffer (600 or 100 μM, respectively), and 39.4 μL was loaded in the syringe. For each binding, three separate experiments were conducted at 25°C (298.15K). Injections were performed with serial injections of peptide; first, one injection of 1 μL, followed by 19 incremental injections of 2 μL, at 120 s intervals. Data from the first injection was excluded, due to pre-equilibration mixing between the contents of cell and syringe at the syringe tip. Peak areas were integrated, normalized, and then fitted by non-linear regression using the independent sites model in Origin™ (version 2.3.6, Microcal, Piscataway, NJ).

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.