Seven-week-old male DBA/1J mice (Orient Bio, Gyeonggi-do, Republic of Korea) were maintained under specific pathogen-free conditions and provided standard laboratory mouse chow (Ralston Purina, St. Louis, MO, USA) and water ad libitum. The mice were housed at five per cage in a room maintained under controlled temperature (21–22°C) and lighting (12/12 h light/dark cycle) conditions. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the School of Medicine and the Animal Research Ethics Committee of the Catholic University of Korea; the procedures complied with the Laboratory Animals Welfare Act, in accordance with the Guide for the Care and Use of Laboratory Animals.
Induction and treatment of arthritis
To induce CIA in DBA/1J mice, chicken type II collagen (4 mg/mL) was dissolved overnight in 0.1 N acetic acid with gentle rotation at 4℃. DBA/1J mice were injected intradermally at the base of the tail with 100 µg of chicken type II collagen; an emulsion of Freund’s adjuvant (Difco, Detroit, MI, USA) was administered to the hind legs of the mice as a booster injection. To assess the eﬀects of L. sakei on the severity of CIA, DBA/1J mice received 50 mg/kg L. sakei in saline, or vehicle alone, via oral gavage six times per week for 7 weeks; this treatment began on day 21 after the primary immunization.
Clinical assessment of arthritis
The severity of arthritis was evaluated by three independent observers. The mice were observed twice weekly to determine the onset and severity of joint inﬂammation for up to 7 weeks after the primary immunization. The severity of arthritis was assessed on a scale of 0–4, based on the following criteria : 0 = no edema or swelling; 1 = slight edema, with erythema limited to the foot or ankle; 2 = slight edema, with erythema from the ankle to the tarsal bone; 3 = moderate edema, with erythema from the ankle to the tarsal bone; and 4 = severe edema, with erythema from the ankle to the entire leg. The arthritis score of each mouse was calculated as the sum of the scores of the four limbs; the highest possible arthritis score for each mouse was 16. The mean arthritis index was used to compare the scores of the control and experimental groups.
Joint tissues were ﬁxed in 10% (v/v) neutral-buﬀered formalin, decalciﬁed in a histological decalcifying agent (Calci-Clear Rapid; National Diagnostics, Atlanta, GA, USA), embedded in paraﬃn, and cut into 5-µm-thick sections. The sections were stained with hematoxylin and eosin, as well as Safranin O, to detect proteoglycans. Inﬂammation was scored using the following criteria: 0 = no inﬂammation; 1 = slight thickening of the lining, or inﬁltration of some cells into the underlying layer; 2 = slight thickening of the lining, with inﬁltration of some cells into the underlying layer; 3 = thickening of the lining, with inﬂux of cells into the underlying layer and cells evident in the synovial space; and 4 = extensive inﬁltration of the synovium by inﬂammatory cells. Cartilage damage was evaluated by staining with Safranin O and toluidine blue, and the extent of damage was scored as follows: 0 = no destruction; 1 = minimal erosion (limited to single spots); 2 = slight-to-moderate erosion in a limited area; 3 = more extensive erosion; and 4 = general destruction.
Immunohistopathological analysis of arthritis
Joint tissue were first incubated with primary antibodies against tumor necrosis factor-α, IL-1β (R&D Systems, Minneapolis, MN, USA),, IL-6(R&D Systems),, and IL-17 (R&D Systems) overnight at 4 °C. Samples were incubated with a biotinylated secondary antibody, followed by incubation with a streptavidin–peroxidase complex for 1 h. Samples were then developed using chromogen 3,3′-diaminobenzidine (Thermo Scientific, Rockford, IL,USA). The sections were examined under a photomicroscope (Olympus, Tokyo, Japan). The number of positive cells was counted using Adobe Photoshop software (Adobe, USA) on high-power digital image (magnification: 400). Positive cells were enumerated visually by three individuals, and the mean values were calculated.
Enzyme-linked immunosorbent assay
The IL-17 and IL-10 concentrations in the supernatants of cultures of human cells were measured by sandwich enzyme-linked immunosorbent assay (ELISA) (R&D Systems). The absorbance at 405 nm was determined using an ELISA microplate reader (Molecular Devices, Sunnyvale, CA, USA).
Isolation and stimulation of splenocytes
Splenocytes were prepared from the spleens of normal C57BL6 mice. Splenocytes were maintained in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 5% fetal bovine serum (Gibco, Grand Island, NY, USA) before stimulation with plate-bound anti-CD3 (0.5 µg/mL) for 3 days; they were then subjected to flow cytometry analysis.
Isolation and stimulation of peripheral blood mononuclear cells
Peripheral blood mononuclear cells (PBMCs) were prepared from heparinized blood by standard Ficoll–Paque density gradient centrifugation (GE Healthcare Biosciences, Uppsala, Sweden). Cells were cultured in RPMI-1640 medium (Gibco BRL, Carlsbad, CA, USA) containing penicillin (100 U/mL), streptomycin (100 μg/mL), and 10% fetal bovine serum (Gibco BRL) that had been inactivated by heating to 55°C for 30 min. Suspensions of both cell types were dispensed into 48-well plates (Nunc, Rosklide, Denmark). PBMCs were incubated with plate bound anti-CD3 (0.5 µg/mL) or lipopolysaccharide 100 ng/mL for 3 days.
Preparation of bacteria
Patients with RA fulfilled the 2010 American College of Rheumatology and European League Against Rheumatism classification criteria . Bacterial genomic DNA isolated from fecal samples of RA patients (RH1114) and healthy normal subjects (RH1117). The study design was approved by the Institutional Review Board of Seoul St. Mary’s Hospital, The Catholic University of Korea (approval ID: KC17TNSI0570). Written informed consents were obtained from all study participants.
50 of each bacterium was inoculated to 5 L cMRS liquid medium and incubated for 20 hours at 37 ℃. After incubation, in order to remove the medium component, the supernatant of the medium was discarded by centrifugation (High Speed Centrifuge, 2236HR, Korea) at 20 ℃ and 7,000 rpm with phosphate buffered saline (PBS) washing, twice. The washed bacterial cells were powdered by drying the remaining medium components at 36 ℃, 2,000 rpm for 24 hours using Scanvac Speed Vacuum Concentrator (Labogene Aps, Lillerød, Denmark). The powdered bacterial cells were heat-inactivated by heating at 80 ℃ for 30min. All bacteria used in this study were isolated from healthy subjects and patients with RA. The information about the donors are presented as supplemental Table 1.
Levels of cytokines and transcription factors were assessed by intracellular staining using anti-IL-17-FITC, anti-Foxp3-FITC, and anti-Foxp3-PE antibodies (all from eBioscience, San Diego, CA, USA). Cells were stimulated with phorbol myristate acetate and ionomycin with the addition of GolgiStop for 4 h. Cultured cells were surface labeled for 30 min and permeabilized with Cytoﬁx/Cytoperm solution (BD Pharmingen, Heidelberg, Germany). Cells were intracellularly stained with ﬂuorescent antibodies and subjected to ﬂow cytometry (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA). Events were collected and analyzed using FlowJo software (Tree Star, Ashland, OR, USA).
In vitro osteoclastogenesis and tartrate-resistant acid phosphatase staining
Bone marrow cells from mouse femurs were cultured in alpha-minimal essential medium (Invitrogen, Carlsbad, CA, USA) containing antibiotics and 10% heat-inactivated fetal bovine serum to separate floating and adherent cells. Nonadherent cells were removed by washing with media, and preosteoclasts were cultured in the presence of 10 ng/mL macrophage colony-stimulating factor, 100 ng/mL receptor activator of nuclear factor kappa-Β (RANK) ligand (RANKL) (PeptoTech, London, UK), and L. sakei for 4 days to generate osteoclasts. The medium was changed every 2 days. Osteoclasts were generated after 8–10 days.
A commercial TRAP kit (Sigma-Aldrich) was used according to the manufacturer’s instructions; however, counterstaining with hematoxylin was not performed. TRAP-positive multinuclear cells (MNCs) containing three or more nuclei were counted as osteoclasts.
Real-time polymerase chain reaction
Polymerase chain reaction was performed using a Light Cycler 2.0 instrument (Roche Diagnostics, Mannheim, Germany) with software version 4.0. All reactions were performed using Light Cycler Fast Start DNA Master SYBR Green I (TaKaRa, Shiga, Japan), in accordance with the manufacturer’s instructions. The following primers were used: TRAP, 5′-TCC TGG CTC AAA AAG CAG TT-3′ (sense) and 5′-ACA TAG CCC ACA CCG TTC TC-3′ (antisense); calcitonin receptor, 5′-CGG ACT TTG ACA CAG AA-3′ (sense) and 5′-AGC AAT CGA CAA GGA GT-3′ (antisense); integrin b3, 5′-CTG TGG GCT TTA AGG ACA GC-3′ (sense) and 5′-GAG GGT CGG TAA TCC TC-3′ (antisense); cathepsin K, 5′-CAG AGG TGT GTA CTA TG-3′ (sense) and 5′-GCG TTG TTC TTA TTC CGA GC-3′ (antisense); RIPK1, 5′-CTG TTC CCT GTG CCC AAT AA-3′ (sense) and 5′-ATG ACT CTG AAG CTG TCC TTT C-3′ (antisense); and RIPK3, 5′-GCA CTC CTC AGA TTC CAC ATA C-3′ (sense) and 5′-GTG TCT TCC ATC TCC CTG ATT C-3′ (antisense).
Statistical analysis was performed using the nonparametric Mann–Whitney U test for comparisons of two groups, and one-way analysis of variance with the Bonferroni post hoc test for multiple groups. Prism ver. 5.01 software (GraphPad Software Inc., San Diego, CA, USA) was used. P < 0.05 was regarded as the threshold for statistical significance. Data are presented as means ± standard deviations.