Male C57BL/6J mice were evaluated at 11–12 weeks of age. All mice used in this experiment were purchased from Charles River Japan Inc. (Yokohama, Japan) and maintained under specific pathogen-free conditions at the Kyushu University Animal Facility. All animal experiments were approved by the Animal Care and Use Committee, Kyushu University.
Induction of collagen-induced arthritis
Mice were immunized subcutaneously at the base of the tail with 4 mg/ml chicken type II collagen (CII) (Collagen Research Center, Tokyo, Japan) emulsified with an equal volume of complete Freund’s adjuvant (CFA) containing Mycobacterium tuberculosis H37Ra at a concentration of 5 mg/ml (Difco, Detroit, MI, USA). Mice were boosted on day 21 with CII emulsified with CFA containing Mycobacterium tuberculosis H37RA at the same concentration.
Measurement of serum anti-type II collagen antibody levels
Serum levels of anti-CII antibodies were measured by enzyme-linked immunosorbent assay (ELISA). Microtiter plates were coated with chicken CII (10 μg/ml) overnight at 4 °C. After washing and blocking, serum samples were added in serial dilutions and incubated 2 h at room temperature. After four washes, HRP-conjugated anti-mouse IgG antibody (Leinco Technologies Inc., St. Louis, MO, USA) was added and incubated for an hour at room temperature. Antibody binding was visualized using TMB substrate (Seracare Life Sciences Inc., Milford, MA, USA). All plates were read at 450 nm using MultiSkan FC (Thermo Fisher Scientific KK, Yokohama, Japan).
Flow cytometric analysis of mouse ILC subsets
To obtain a cell suspension from mouse joints, the entire legs were dissected and the muscles and tendons were removed. To avoid contamination with the bone marrow, the femur was disarticulated by pulling the femoral head. The ligaments and tendons around the joints were cut a few millimeters. The knee and ankle joint were then opened. The legs were incubated in a mixture of enzymes containing 100 U/ml collagenase (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and 100 μg/ml DNaseI (Sigma-Aldrich, St. Louis, MO, USA) and shaken for 60 min at 37 °C. After digestion, the legs were removed, and cells were purified using Percoll density centrifugation, filtered, and counted using automated cell counter.
The popliteal lymph nodes and spleen were mechanically mashed and filtered. Splenocytes and peripheral blood cells were hemolyzed with NH4Cl and washed with PBS. For surface staining, single-cell suspensions of joint cells, lymph node cells, splenocytes, and peripheral blood cells were washed with cell staining buffer and stained with a cocktail of antibodies for 30 min at 4 °C. ILCs were defined as CD45.2 positive, lineage (CD3, CD8, CD19, B220, Ter119, Ly-6G, CD11c, CD11b) negative, CD90.2 (Thy1.2) positive, and CD127 (IL-7R) positive. ILCs were subsequently classified as ILC1s, ILC2s, CCR6+ ILC3s, or NKp46 (CD335)-positive ILCs (NKp46+ ILCs), based on the expression of NK1.1, ST2 (the receptor for IL-33), CCR6, and NKp46.
RNA extraction and quantitative real-time PCR
Each target population was sorted directly into ISOGEN II (Nippon Gene Co., Tokyo, Japan), and total RNA was extracted and reverse transcribed using the SuperScript III First-Strand Synthesis System (Invitrogen). Quantitative real-time PCR was conducted with a 7500 Real-Time PCR System (Applied Biosystems, Foster, CA, USA) or an Mx 3000P Real-Time PCR System (Agilent Technologies, Santa Clara, CA, USA). TaqMan probes for Tbx21 (Mm00450960_m1), Gata3 (Mm00484683_m1), RORC (Mm01261022_m1), Ifng (Mm01168134_m1), Il5 (Mm00439646_m1), Il13 (Mm99999190_m1), Il17Aa (Mm00439618_m1), and Il22 (Mm00444241_m1) were all purchased from Applied Biosystems. 18S rRNA expression served as the internal control.
Relative transcript levels were calculated using the ddCT method.
Patients with RA met the 1987 American College of Rheumatology classification criteria. Twenty-three synovial fluid (SF) samples were obtained from 17 patients with RA. Patient details are provided in Additional file 8: Table S2. Twenty-four SF samples of patients with osteoarthritis (OA) were obtained from 20 patients. Informed consent was obtained from all subjects in accordance with the Declaration of Helsinki. The Institutional Review Board of Kyushu University Hospital approved all research on human subjects.
Flow cytometric analysis of ILCs in synovial fluid in RA patients
Mononuclear cells were isolated from SF using density gradient centrifugation with LSM (MP Biomedicals, LLC, Santa Ana, CA, USA). ILCs were defined as single cells within the lymphocyte gate on the scatter plot that were CD45 positive, lineage (CD3, CD19, CD16, CD94, CD14, CD1c, CD11b, CD11c, CD235ab, FcεRI, CD34) negative, and CD127 (IL-7R) positive. ILC subsets were classified based on the expression of CD117 (c-Kit), chemoattractant receptor homologous molecule expressed on Th2 cells (CRTH2) (CD294), and NKp44 (CD336). Specifically, ILC2s were defined as CRTH2 positive, and other subsets were CRTH2 negative. ILC1s, NKp44− ILC3s, and NKp44+ ILC3s were defined as CD117− NKp44−, CD117+ NKp44−, and CD117+ NKp44+, respectively. CCR6+ ILCs were defined as CCR6-positive cells in total ILCs. Antibodies used in flow cytometric analysis are listed in Additional file 7: Table S1 and were purchased from BD Biosciences (San Jose, CA, USA), BioLegend (San Diego, CA, USA), Beckman Coulter (Villepinte, France), or MD Bioproducts (Saint Paul, MN, USA). Cells were analyzed with a FACS Aria III system (BD Biosciences). FlowJo software (Tree Star Inc., Ashland, OR, USA) was used to analyze flow cytometric data.
SF from RA and OA patients was centrifuged, and supernatants were collected and stored at − 80 °C. Human CC chemokine ligand 20 (CCL20) in SF was measured by ELISA using a Human CCL20 ELISA kit (BioLegend) with a detection limit of 2.9 pg/ml, according to the manufacturer’s instructions.
Statistical comparisons between the two groups were performed using Student’s t test or the Wilcoxon rank-sum test according to the distributions. Results are expressed as means ± SD or the median (range). Differences between the three groups were assessed by the Kruskal-Wallis test followed by the Wilcoxon rank-sum test. Spearman’s rank correlation was performed to correlate the proportion of ILC subsets in SF with clinical parameters of RA patients. P values < 0.05 were considered significant. All analyses were performed using JMP statistical software version 13.0 (SAS Institute, Cary, NC, USA).