Mice
Male DBA/1 mice were purchased from Charles River Japan (Tokyo, Japan), and used at 6–10 weeks of age. All mice were maintained under specific pathogen-free conditions at the University of Tsukuba. All experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Tsukuba, and all animal experiments were conducted in accordance with institutional ethics guidelines. All surgeries were performed under isoflurane anesthesia, and utmost care was taken to minimize suffering.
Serum samples
Serum samples were collected from Japanese patients with RA (n = 60, mean age 52.2 years, range 20–73 years, females 80%) diagnosed by rheumatologists according to the 1987 American College of Rheumatology (ACR) classification criteria [14] or the 2010 ACR/European League Against Rheumatism classification criteria [15]. Serum samples of the disease control subjects were collected from Japanese patients with primary Sjögren’s syndrome (SS) (n = 27, mean age 58.8 years, range 26–80 years, females 96%), systemic lupus erythematosus (SLE) (n = 15, mean age 33.9 years, range 16–55 years, females 80%), or osteoarthritis (OA) (n = 12, mean age 56.3 years, range 37–80 years, females 67%). All the patients with SS were diagnosed by rheumatologists according to the 1999 Japanese Ministry of Health criteria for diagnosis of SS [16]. All the patients with SLE fulfilled the 1997 ACR classification criteria [17]. None of the patients with SS or SLE had overlapping RA. Serum samples were collected from healthy subjects (HS) (n = 30, mean age 49.0 years, range 34–65 years, females 80%). Serum samples were also collected from 17 patients with RA before and 24 weeks after treatment with biologic drugs (infliximab, n = 9; abatacept, n = 8). All samples were collected at the University of Tsukuba Hospital after informed consent was obtained. This study was reviewed and approved by the ethics committee of the University of Tsukuba.
Peptide GPI-induced arthritis
DBA/1 mice were immunized with 25 μg of pGPI (Invitrogen/Thermo Fisher Scientific, Carlsbad, CA, USA) in complete Freund’s adjuvant (CFA) (BD Biosciences, San Jose, CA, USA). pGPI was emulsified with CFA at a 1:1 ratio (vol/vol), or PBS + CFA was prepared as a vehicle control. For induction of arthritis, 150 μl of the emulsion was injected intradermally at the base of the tails of the mice. Each mouse was also given an injection of 200 ng of pertussis toxin (Sigma-Aldrich, St. Louis, MO, USA) intraperitoneally on days 0 and 2 after immunization to induce arthritis. Arthritis was assessed every other day and evaluated using a scale of 0–3 for swelling and redness of each paw. The clinical score was the sum of the scores for four paws, as described previously [11].
Measurement of anti-CCP antibodies in pGIA
Sera were obtained on days 0–28 every week from mice immunized with pGPI or control, and antibodies were measured by enzyme-linked immunosorbent assay (ELISA). Sera were diluted 1:25 in dilution buffer and added to the 96-well plate (Immunoscan CCPlus test kit; Euro Diagnostica, Malmö, Sweden) for 1 h at room temperature. After a washing step, horseradish peroxidase (HRP)-conjugated polyclonal rabbit antimouse immunoglobulin (Dako, Carpinteria, CA, USA) diluted 1:1000 was added for 30 minutes at room temperature. After another washing step, color was developed with 3,3′,5,5′-tetramethylbenzidine (TMB) microwell peroxidase substrate (KPL/SeraCare, Milford, MA, USA). The optical density (OD) was measured at 450 nm by using a microplate reader.
A standard pool was obtained by mixing sera obtained from several mice on day 28. The concentrations of antibodies in this pool were considered 100 U/ml. A standard curve was obtained using serum dilutions, and the Michaelis-Menten equation was used to convert OD values into units, as described previously [18].
Measurement of anti-ITIH4 antibodies in patients with RA
Native peptide ITIH4428–447 and R438 citrullinated peptide ITIH4428–447 were synthesized (serum, purity 95%) and used in ELISA. Ninety-six-well plates (Nunc MaxiSorp; Thermo Fisher Scientific) were coated with 10 μg/ml peptides for 12 h at 4 °C. After washing and blocking steps, sera from patients with RA (n = 60) and HS (n = 30) were diluted 1:200 in 1% bovine serum albumin (BSA) in PBS, then added for 2 h at room temperature. After a washing step, HRP-conjugated goat antihuman immunoglobulin G (IgG) (heavy and light chains [H + L]) (Abcam, Cambridge, MA, USA) diluted 1:10,000 was added for 1 h at room temperature. After washing, color was developed with TMB microwell peroxidase substrate (KPL/SeraCare). The OD was measured at 450 nm by using a microplate reader. The cutoff value was determined as the mean + 2 SD of HS.
Histopathological analysis
Ankle joint tissue samples were harvested at days 0 and 14 from mice immunized with pGPI or control. They were fixed in neutralized 10% formalin, embedded in paraffin, and sectioned. To detect citrullinated proteins, we prepared modification buffer by mixing Reagent A (20% H2SO4, 25% H3PO4, and 0.025% FeCl3) and Reagent B (1% diacetyl monoxime, 0.5% antipyrine, 1 M acetic acid) at a 2:1 ratio (vol/vol). The sections were covered with the modification buffer and incubated in a light-proof container at 37 °C for 2.5 h to modify citrulline residues. Then, the sections were incubated overnight at room temperature with rabbit anti-modified citrulline (AMC) polyclonal antibodies diluted 1:3200 in 2% BSA in PBS to detect modified citrulline residues [19]. The sections were also incubated with HRP-conjugated goat antirabbit IgG (H + L) (Bio-Rad Laboratories, Hercules, CA, USA) for 30 minutes at room temperature. The sections were also stained with 3,3′-diaminobenzidine (DAB) (Nichirei Biosciences, Tokyo, Japan) and hematoxylin. To detect ITIH4 or macrophages, IHC analysis of the joint or liver (as a positive control of ITIH4 staining) sections was performed as described above, using rabbit antihuman ITIH4 antibodies (Abcam) diluted 1:1000 and rat antimouse F4/80 antibodies (BioLegend, San Diego, CA, USA) diluted 1:200 as primary antibodies and using rabbit-specific IHC polymer detection kit HRP/DAB (Abcam) and HRP-conjugated polyclonal rabbit antirat immunoglobulin (Dako) diluted 1:200 as secondary antibodies, respectively.
Real-time qPCR analysis
We extracted total RNA from the ankle joints of pGIA or control mice using the ISOGEN (Wako Pure Chemical Industries, Tokyo, Japan) extraction method according to the instructions provided by the manufacturer. The extracted RNA was reverse-transcribed to complementary DNA with random primers. We performed real-time qPCR using a TaqMan gene expression assay (Applied Biosystems/Thermo Fisher Scientific, Foster City, CA, USA), and the Padi4 (Mm01341658_m1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (NM_002046) primers. Real-time qPCR was carried out using an ABI 7500 analyzer (Applied Biosystems). The expression of GAPDH was used as the control.
Western blot analysis
Serum samples were obtained at days 0, 7, 14, and 28 from mice immunized with pGPI or control, as well as from patients with RA, SS, SLE, or OA and from HS. For analysis of citrullinated protein expression, serum samples were loaded into each well (50 μg for SDS-PAGE or 100 μg for two-dimensional PAGE [2D-PAGE]), separated by SDS-PAGE or 2D-PAGE, and transferred to polyvinylidene difluoride membranes. The modification buffer was added to the blots before incubation in a light-proof container at 37 °C for 2.5 h to modify citrulline residues, as described above. Blots were washed with 0.05% Tween 20 in Tris-buffered saline (TBST) and blocked with 5% milk in TBST, then incubated overnight at 4 °C with AMC antibodies diluted 1:3200 in 5% milk in TBST [19]. After a washing step, the blots were incubated with secondary antibody HRP-conjugated goat antirabbit IgG (H + L) (Bio-Rad Laboratories) diluted 1:5000 in 5% milk in TBST for 1 h at room temperature. Densitometric analysis was carried out using an ImageQuant LAS 4000 densitometer (GE Healthcare Life Sciences, Marlborough, MA, USA). The band intensity was determined with ImageQuant TL software (GE Healthcare Life Sciences) and normalized using the value of one sample (pGIA or RA) as 10. The difference of citrullinated ITIH4 levels from baseline to 24 weeks after treatment was normalized using each value at baseline. For analysis of ITIH4 expression, serum samples were loaded 10 μg/well and Western blot analysis was performed as described above, using rabbit antihuman ITIH4 antibodies (Abcam) diluted 1:3000.
Measurement of citrulline content
Sera were obtained at days 0, 7, 14, and 28 from mice immunized with pGPI or control, and citrulline contents were measured by using the color development reagent (COLDER) assay, as described previously [20]. Sera were diluted 1:100 in COLDER buffer (50 mM NaCl, 10 mM CaCl2, 2 mM dithiothreitol, and 100 mM Tris, pH 7.4), and 60 μl of samples were added to the 96-well plates (Nunc MaxiSorp). Then, 200 μl of COLDER were added to detect citrullinated proteins. The samples were incubated for 30 minutes at 95 °C. The OD was measured at 570 nm by using a microplate reader and compared with a standard curve of known citrulline concentrations. Data were normalized relative to the protein concentration in each sample.
LC-MS
Sera from pGIA mice, control mice, patients with RA, and HS were separated by 2D-PAGE, and gels were stained with GelCode Blue Stain Reagent (Thermo Fisher Scientific) for Coomassie brilliant blue staining. Gel slices were incubated overnight with MS-grade modified trypsin (6.7 ng/μl; Promega, Madison, WI, USA) at 37 °C. The resultant peptides were analyzed with the nanoACQUITY ultrahigh-performance LC (UPLC) system (Waters, Milford, MA, USA). Data were collected in centroid mode from mass-to-charge ratios (m/z) 50 to 1990. All analyses were acquired with an independent reference. BiopharmLynx version 1.2 software (Waters) was used for baseline subtraction and smoothing, de-isotoping, de novo peptide sequence identification, and database searches.
Statistical analysis
All data were expressed as mean ± SEM. Differences between groups were evaluated for statistical significance using Student’s t test. The Kruskal-Wallis test was used for evaluation of band intensity among the five subject groups. P values less than 0.05 were considered significant. Statistical analyses were performed using IBM SPSS Statistics software (IBM, Armonk, NY, USA).