Preparation of bacteria
Bacteria used in this study were purchased from ATCC. Pg W83 and 33277 were cultured on a sheep blood agar plate using the Anaeropack system (Mitsubishi Gas Chemical, Tokyo, Japan) at 37°C. After a 2-day incubation, Pg was inoculated in 40 mL of trypticase soy broth supplemented with 1% yeast extract, hemin (200 mg), and menadion (20 mg). Bacteria were harvested in the exponential growth phase and washed with phosphate buffered saline (PBS) for experiments.
Construction of Pg knockout mutant
The Pg pad knockout mutant was constructed by double recombination of the target gene and ermF introduction, as previously described . The targeting DNA was constructed as follows. The 0.5 kb-upstream and 0.5 kb-downstream regions of the pad gene were amplified with two pairs of primers (pad-Up-F: [GGTCTCACACGAGAGGATACTATGGTCTAT]/pad-Up-R: [CGGGGGATCCTGTTTGATATGTTTTATGAT]; pad-Dw-F: [TAGGGGATCCGGGGCCTTATTTGAGAATAC]/pad-Dw-R: [AGCAGAGGTTACGAGCTTAACCAGAGATGC], where ‘Up’, ‘Dw’, ‘F’, and ‘R’ indicate upstream, downstream, forward, and reverse, respectively) using the genome of ATCC 33277 as a template. The ermF region in the ermF DNA cassette was amplified with ermF-F: [ATATCAAACAGGATCCCCCGATAGCTTCCG]/ermF-R: [ATAAGGCCCCGGATCCCCTACGAAGGATGA] using the genome of gtfF (PGN_1668)::ermF mutant (KDP611) as a template . Using the three purified products, PCR was performed with pad-Up-F/ pad-Dw-R. Finally, the desired PCR product was purified and introduced into Pg ATCC 33277 by electroporation. Transformants were selected on blood agar plates containing 10 μg/mL erythromycin. Proper mutation was confirmed by PCR using pad-Up-R/pad-Dw-R. Correct ermF gene insertion of Pg pad knockout mutant was also verified by sequencing.
Generation of Pg-infected experimental arthritis mouse model
All animal experimental procedures employed in this study were approved by the Ethical Committee of Hiroshima University (approved No. A16-33) and performed as previously reported . Briefly, SKG mice were maintained under SPF condition until inoculation of bacteria and feces. The food used in this study was MF for mouse with gamma irradiation (Oriental Yeast Co., Ltd, Tokyo, Japan). Laminarin derived from Laminaria digitate (LA) was purchased from Sigma-Aldrich (L9634, St. Louis, MO, USA). LA was dissolved in PBS at 100 mg/mL. To induce experimental arthritis, LA (10 mg/100 mL/mouse) was administered to SKG mice by an intraperitoneal (i.p.) injection. Pg was also inoculated (108 bacterial cells/50 mL in 2% carboxymethylcellulose (CMC) solution) twice a week for 6 weeks. LA injection and Pg inoculation were started the same day. The same volume of CMC solution was inoculated as a negative control. All mice were divided into 3–4 groups (5 or 6 mice per group) in each experiments (Ctrl: CMC inoculation, LA: LA i.p injection, Pg: Pg oral inoculation, Pg/LA: LA i.p. injection with Pg oral inoculation, LA-FMT: LA i.p. injection with FMT from the LA group, Pg/LA-FMT: LA i.p. injection with FMT from the Pg/LA group).
Evaluation of alveolar bone level in mouse
The alveolar bone level (ABL) of SKG mice was evaluated by the Kawai’s methods described previously . Briefly, after methylene blue staining (Sigma-Aldrich) for 10 min, the upper molar jaw was washed with PBS three times. The length of the blue-stained root surface of all molar teeth from the enamel-cement junction to the top of the alveolar bone was measured. Differences between treated mice and the control group (no treatment) were evaluated.
Clinical assessment of arthritis score (AS) in SKG mice
Joint swelling was monitored by inspection and scored as follows: 0, no joint swelling; 0.1, swelling of one finger joint; 0.5, mild swelling of the wrist or ankle; 1.0, severe swelling of the wrist or ankle. Scores for all digits, wrists, and ankles were totaled for each mouse, as reported previously .
IL-6 measurement in mouse tissue
Tissues from gingiva, leg joint, small intestine, large intestine, and serum were collected from each mouse and homogenized by cool-mill (#TK-CM20S, Tokken, Inc., Chiba, Japan) in the RIPA Lysis and Extraction Buffer (#Thermo Fisher Scientific, Tokyo, Japan, 100 mg tissue/100 mL) with 0.1% phenylmethanesulfonyl fluoride (PMSF, Sigma-Aldrich) and 1% proteinase inhibitor cocktail (#87786, Thermo Fisher Scientific). The sera were diluted four times by PBS. Supernatants of the tissue homogenates and sera were used for IL-6 measurement by ELISA (for mouse IL-6, #431304; BioLegend Inc., San Diego, CA, USA), according to the manufacturer’s instructions. Briefly, a solid-phase anti-IL-6 monoclonal antibody (diluted in coating buffer to a final concentration of 1 μg/mL) was coated onto a 96-well ELISA plate (BD Falcon, Franklin Lakes, NJ, USA) for target capture. After blocking each well with 1% BSA in PBS supplemented with 0.05% Tween 20 (PBST), the sample or standard (diluted in PBST from 1 ng/mL to zero) was applied to each well. After application of the detection antibody (diluted in PBST to a final concentration of 1 μg/mL), horseradish peroxidase (HRP) conjugated with anti-IgG (2000-fold dilution in PBST) was applied to the wells. Colorimetric reactions were developed with o-phenylenediamine (Sigma-Aldrich) in the presence of 0.02% H2O2. Color development was stopped with H2SO4 (2N) and measured using an ELISA reader (OD405, Varioskan LUX). The actual concentration of the target was calibrated by referring to a standard curve prepared by serial dilutions. Each sample was examined in triplicate wells of a 96-well ELISA plate. The limits of mouse IL-6 detection for each analyte were 15.6 pg/mL.
Detection of citrullinated protein in mouse tissue
The homogenized tissues from the gingiva, leg joint, small intestine, large intestine, and serum were diluted in sodium bicarbonate buffer (pH 9.4, 10 mg/mL) and coated onto a 96-well ELISA plate. After blocking each well with 1% BSA and sucrose in PBST, anti-citrulline monoclonal IgG (CCP-Ab1 generated from B cells of an RA patient, 10 μg/mL) in PBST at room temperature for 2 h . Subsequently, anti-human IgG conjugated with HRP (2000-fold dilution in PBST) was applied to the wells. Colorimetric reactions were performed as the same as the method of IL-6 measurement.
Measurement of serum ACPA level
Serum from each group was collected to measure ACPA levels. Briefly, cyclic citrullinated peptides (CCP) (Orgentec, Chicago, IL, USA, diluted in PBS pH 7.2 at final concentration of 0.5 μg/mL) was pre-coated onto a 96-well ELISA plate (BD Falcon, Franklin Lakes, NJ, USA) for target capture. After blocking each well with 1% BSA in PBST, the serum (4-fold dilution) or standard (diluted in PBST from 1 ng/mL to zero) was applied to each well. After application of the detection antibody (diluted in PBST to a final concentration of 1 μg/mL), anti-IgG conjugated with HRP (2000-fold dilution in PBST) was applied to the wells. Colorimetric reactions were developed using the same protocol as IL-6 detection. The actual concentration of the target was calibrated by referring to a standard curve prepared by serial dilutions. Each sample was examined in triplicate wells of a 96-well ELISA plate.
Ankle joints were fixed in 4% buffered formalin and embedded in paraffin wax. Subsequently, the tissues were sliced at a thickness of 7 μm and mounted on glass slides. The paraffin-embedded sections were stained with hematoxylin and eosin (HE). The severity of inflammation and cartilage damage were scored using published criteria .
All experiments were performed at least three times independently. Data are expressed as mean ± standard deviation (SD). Statistical analyses between two groups were performed using Mann-Whitney U test for non-normal distribution. For multiple comparisons, the Tukey-Kramer test or Bonferroni-corrected Mann-Whitney U test was used. P < 0.05 was considered significant.
Metagenomics of gut microbiota
Bacterial DNA was extracted from feces as described previously . In brief, feces were collected from all the group of mice before Pg inoculation (day 0) and at the end of experiments (day 42). The collected feces were suspended in PBS (10% sodium dodecyl sulfate, 10 mM Tris-HCl, and 1 mM EDTA, pH 8.0), and the bacterial DNA was purified using the DNeasy PowerSoil Kit according to the manufacturer’s instructions (the range of total amount of DNA 349.8 ng/ml to 611.8 ng/ml, #12888-100 QIAGEN, Germantown, MD, USA). Following, the V4 variable region (515F–806R) of the bacterial 16S rRNA genes was sequenced on an Illumina Miseq, as the illumine 16s metagenomics sequencing workflow and previously described . Each reaction mixture contained 15 pmol of each primer (16S Amplicon PCR Forward Primer 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG, 16S Amplicon PCR Reverse Primer 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC), KAPA HiFi HotStart ReadyMix (x2) (Hokkaido System Science Co., Ltd., Sapporo, Japan), 50 ng extracted DNA, and sterilized water to reach a final volume of 50 μL. PCR conditions were as follows: 95°C for 3 min; 25 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 min; 72 °C for 5 min. The PCR product was purified by AMPure XP (Beckman Coulter, Inc., Brea, CA, USA) and quantified using a Quant-iT PicoGreen ds DNA Assay Kit (Life Technologies Japan Ltd., Tokyo, Japan). Mixed samples were prepared by pooling approximately equal amounts of PCR amplicons from each sample. The pooled library was analyzed with an Agilent High Sensitivity DNA Kit on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Real-time PCR was performed on the pooled library using a KAPA Library Quantification Kit for Illumina, following the manufacturer’s protocols, and a sample library with a 20% denatured PhiX spike-in was sequenced by Miseq using a 600 cycles kit to obtain 2 × 250 bp paired-end reads. Taxonomic assignments and estimation of relative abundance of sequencing data were performed using the analysis pipeline of the QIIME software package . An operational taxonomic unit (OTU) was defined at 97% similarity. OTUs indicating relative abundance under 0.05% were filtered to eliminate noise. The OTU was assigned a taxonomy based on a comparison with the Silva database using UCLUST .
Fecal microbiota transmission (FMT)
Fresh mouse feces from the LA and Pg/LA group were collected after 6 weeks of Pg inoculation started and mixed from all the same group (n=6), and homogenized in anaerobic resuspension buffer (2% Lab–Lemco powder, 0.1% L-cysteine, 0.045% KH2PO4, 0.09% NaCl, 0.045% (NH4)2SO4, 0.0045% CaCl2, 0.0045% MgSO4 and 40% glycerol in 1,000 mL) at 10-fold dilution (weight/volume) and kept at −80°C until used. Before FMT, SKG mice were treated with antibiotics (ampicillin (Sigma-Aldrich) 1 g/L,metronidazole (Sigma-Aldrich) 1 g/L, neomycin (Sigma-Aldrich) 1g/L, vancomycin (Sigma-Aldrich) 0.5 g/L) dissolved in water for 1 week. Female SKG mice (6–8 weeks old) were orally inoculated with 250 μL of the fecal suspensions from donor mice twice in a week. Following, these mice were intraperitoneally injected with LA (100 mg) at 1 week after bacterial inoculation. The AS and ankle thickness were monitored every week for 6 weeks after injection. Microbiota composition of the feces from the donor mice (the group of LA and Pg/LA mice after 6 weeks of LA injection with or without Pg inoculation) and the recipient mice at 6 weeks after FMT was analyzed by next generation DNA sequencing of bacterial 16s rRNA. These experiments were performed 3 times independently.
Homoginized tissues from the leg joint were applied to a 12% SDS-polyacrylamide gel by for electrophoresis and the electronically transferred onto nitrocellulose membranes (BioRad Laboratories, CA, USA). The membrane was blocked with 1% non-fat dried milk at room temperature for 1 hour and then reacted with anti-PADI4 rabbit monoclonal IgG (10 μg/ml, EPR20706, abcam, Japan) in PBST at 4°C for 12 hours. The membrane was incubated with HRP-conjugated with sheep anti-rabbit IgG in PBST at room temperature for 1 hour. Immunodetection was performed according to the manual supplied with ECL Plus Western blotting reagents (GE Healthcare Life Sciences, Japan). As a control, the amount of b-actin was detected by anti-b-actin antibody (10 μg/ml, sc-47778 HRP, Santa Cruz Biotechnology, Inc.) The density of target PADI4 bands were measured by NIH image software.