Biological samples were obtained under a protocol approved by the Institutional Research Ethics Committee (IREC) of Nazarbayev University, Astana, Kazakhstan. All subjects gave written informed consent. Patients coming to outpatient facility of the Republican Diagnostics Centre (RDC, Astana, Kazakhstan), who presented with clinically apparent synovial swelling were examined for RA symptoms. The final diagnostic outcome was based on persistent inflammatory arthritis, magnetic resonance imaging (MRI), radiographic analysis, disease duration, and number of tender and swollen joints, and resulted in the 28-joint-count disease activity score (DAS28). Clinical assessment was accompanied with peripheral blood analysis for complete blood counts with differential, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), rheumatoid factor (RF), and anti-citrullinated protein antibodies (ACPA). Venous blood was collected into heparinized tubes, and cells were removed by centrifugation at 1000 × g for 10 minutes. Plasma was stored at –80 °C.
ELISA for CTHRC1
Sandwich ELISA was used to quantify CTHRC1 in human plasma according to the manufacturer’s protocol (www.mmcri.org/antibody, Maine Medical Center Research Institute, Scarborough, ME, USA). Briefly, 96-well plates (Maxisorp, Nunc) were coated overnight at 4 °C with capture antibody 13E09 at 1.8 μg/ml in carbonate-bicarbonate buffer pH 9.4. All subsequent procedures were performed at room temperature. The next day, wells were washed twice with PBS containing 0.1 % BSA and 0.1 % Tween 20 (buffer PBS-BT) and then blocked with PBS-BT for 1 hour. Human plasma or conditioned media were diluted at least 1:5 in PBS-BT and incubated with absorbed capture antibodies for 2 hours. Subsequently, the wells were washed and then incubated with biotinylated detection antibody Vli10G07 diluted 1:500 in PBS-BT for 1 hour. After washing, wells were treated for 1 hour with streptavidin conjugated with horseradish peroxidase (St-HRP) (Pierce High Sensitivity St-HRP, Thermo Fisher Scientific Inc., Waltham, MA, USA) diluted at 1:8,000 in PBS-BT. After the final wash, TMB (3,3′,5,5′-tetramethylbenzidine) chromogenic substrate (Amresco, Solon, OH, USA) was added, and the developed signal was measured at 450 nm using the Multiscan-FC plate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA). Absorbance was converted to absolute concentration using rhCTHRC1 as a reference. ELISA was performed in triplicates.
Animals and arthritis induction
Mice were housed in a specific pathogen-free environment in the Institute for Animal Studies at the Albert Einstein College of Medicine, Bronx, NY, USA. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Albert Einstein College of Medicine. Inbred BALB/c female mice, 2–4 months old (Charles River, Wilmington, MA, USA) were injected with ArthritoMab antibody cocktail (MD Biosciences Inc., St.Paul, MN, USA) according to the manufacturer’s protocol. Briefly, mice were injected intraperitoneally (i.p.) with 2 mg/mouse of antibodies on day 0 followed by i.p. injection of 40 μg of lipopolysaccharide (LPS) on day 3. Arthritis in the (FVB/N × BALB/c) F1 hybrid mice was induced similarly, but using CIA-MAB-2C ArthritoMab Cocktail (MD Biosciences Inc.), which was developed for murine strains that are refractory to arthritis. Mice were observed once or twice a day for paw swelling and redness by two independent observers. The arthritis scoring system was based on the number of inflamed joints in each paw producing a total score of 0–60 per mouse, as described previously [9, 17].
Total RNA was isolated from mouse paws using the RNeasy Mini kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer’s instructions. Paw tissue was taken from digits to the ankle or to the distal radius/ulna. RNA integrity and quality was tested on an Agilent 2100 Bioanalyzer. The Superscript First-Strand Synthesis kit (Invitrogen Life Technologies, Grand Island, NY, USA) was used to generate cDNA that was assayed using an Applied Biosystems 7900HT Fast Real-Time PCR System. Expression was measured in triplicates and at least two repeats using the Fast SYBR Green system (Applied Biosystems by Life Technologies, Grand Island, NY, USA). Cthrc1-specific primers were forward 5′-GGGATGGATTCAAAGGGGAAA-3′ and reverse 5′-AGAACTCGCAGAGCACTGTT-3′. Gapdh-specific primers were forward 5′-ACCCAGAAGACTGTGGATGG-3′ and reverse 5′-ACACATTGGGGGTAGGAACA-3′. Relative RNA concentration was calculated using the delta-delta cycle threshold (Ct) method corrected for PCR efficiency, as described previously [9, 18].
Human RA-FLS derived from the inflamed synovial tissue of patients with RA (Cell Applications Inc., San Diego, CA, USA) were used at passages three to four. RA-FLS were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with GlutaMAX-1, 4.5 g/l D-glucose, and 25 mM HEPES, antibiotic and antimycotic (Gibco by Life Technologies, Grand Island, NY, USA) supplemented with 10 % heat-inactivated FBS and growth supplements (Cell Applications Inc., San Diego, CA, USA). Mouse embryonic fibroblast cell lines C3H/10T1/2 and NIH 3T3, and human skin fibroblast line Hs68 (all lines from ATCC Inc., Manassas, VA, USA) were studied.
Histopathology and immunohistochemical analysis (IHC)
Mice were killed by CO2 inhalation according to the approved IACUC protocol. For histopathological analysis, hind limbs from the digits to femur, including the knee joint, were collected. Tissues were fixed in 10 % neutral buffered formalin, decalcified using Immunocal reagent (Decal Chemical Corporation, Suffern, NY, USA) and embedded in paraffin. Tissues were sectioned at a thickness of 5 μm and stained with hematoxylin-eosin or alcian blue and nuclear fast red for histological confirmation of arthritis.
For immunohistochemical analysis, sections were deparaffinized and pretreated with 10 mM sodium citrate buffer at pH 6.0 heated to 96 °C for 20 minutes for antigen retrieval. Endogenous peroxidase activity was quenched by incubation in 0.3 % hydrogen peroxide in PBS for 5 minutes. Nonspecific binding was blocked for 1 hour at room temperature using 2 % BSA (Sigma-Aldrich, St. Louis, MO, USA) and 2 % normal goat serum (Vector laboratories Inc., Burlingame, CA, USA) in PBS and 0.05 % Tween-20 (PBS-T buffer). For detection of FLS-specific and macrophage-specific markers in arthritic pannus, we used rabbit anti-CTHRC1 antibodies (Vli-55, www.mmcri.org/antibody, Scarborough, ME, USA), rabbit anti-IBA1 antibodies (#019-19741, Wako Chemicals USA Inc., Richmond, VA, USA), rabbit anti-CDH11 antibodies (WTID1, Invitrogen by Life Technologies, Grand Island, NY, USA), and rabbit antibodies to smooth muscle actin (α-SMA) (E184, Abcam, Cambridge, MA, USA). As a secondary antibody, donkey anti-rabbit antibodies conjugated to HRP (Abcam, Cambridge, MA, USA) were used. After three washes with PBST, slides were finally stained with ImmPact 3,3′-diaminobenzidine (DAB) and counterstained with hematoxylin (Vector laboratories Inc.) or alcian blue (Thermo Fisher Scientific Inc., Waltham, MA, USA). Sections were dehydrated and mounted with Permount (Thermo Fisher Scientific Inc.).
Recombinant protein overexpression
We used a full-length human Cthrc1 cDNA cloned in frame with turbo-GFP (clone RG204348, OriGene Technologies, Inc., Rockville, MD, USA) as a template to amplify a Cthrc1 sequence. High fidelity PCR using Herculase II fusion DNA polymerase (Agilent Technologies, Inc., Santa Clara, CA, USA) with forward 5′-ATTTGGTACC
ATGCGACCCCAGGGCCCCGCCG-3′ and reverse 5′-AATAGCGGCCG
ATGATGATGATGATGATGTTTTGGTAGTTCTTCAAT-3′ primers/linkers to introduce sites for restriction endonucleases Kpn°I and Hind°III and 6xHis tag and stop codon (restriction sites are underlined; ATG and stop codons are boldfaced; 6xHis coding sequence is italicized and underlined) for cloning the amplicon into the pCMV6-K/N vector (OriGene Technologies, Inc.). The recombinant construct structure was confirmed with a complete sequencing (Thermo Fisher Scientific Inc., Fair Lawn, NJ, USA).
Transient transfection of the Chinese hamster ovary (CHO) cells with lipofectamine LFA2000 was performed according to the manufacturer’s protocol (Life Technologies, Grand Island, NY, USA). CHO cells were grown in Opti-CHO chemically-defined serum-free conditioned media Opti-CHO (Gibco Life Technologies, Grand Island, NY, USA) for 5 days. Conditioned media were collected and centrifuged at 1000 × g for 15 minutes to separate from floating cells.
Cells were lysed in Laemmli sample buffer (62.5 mM Tris-HCl, 2 % sodium dodecyl sulfate (SDS), 10 % glycerol, 0.05 % bromphenol blue, and 100 mM 2-mercaptoethanol). Whole cell/tissue lysates were separated on 4–15 % gradient SDS-polyacrylamide gels (Bio-Rad Laboratories Inc., Hercules, CA, USA) and transferred onto polyvinylidene fluoride membrane (Thermo Fisher Scientific Inc., Waltham, MA, USA). Membranes were blocked for 1 hour in 1 % (w/v) casein in 25 mM Tris pH 7.4, 150 mM NaCl, 0.1 % Tween 20 (TBS-T) (casein blocking buffer, Thermo Fisher Scientific Inc.). After blocking, the membranes were probed with rabbit antibodies to CTHRC1 (Vli-55, www.mmcri.org/antibody, Scarborough, ME, USA), rabbit antibodies to CDH11 (Invitrogen Life Technologies, Grand Island, NY, USA), rabbit antibodies to α-SMA (Abcam, Cambridge, MA, USA), mouse antibodies to α-tubulin (Sigma-Aldrich, St. Louis, MO, USA) as protein load control. As secondary antibodies, HRP-conjugated donkey anti-rabbit, anti-mouse or anti-sheep antibodies (Abcam) were used. Enhanced chemiluminescent substrate (Thermo Fisher Scientific Inc.) and HyBlot-CL film (Denville Scientific Inc., Holliston, MA, USA) were used to detect a signal. Optical density was quantified by ImageJ analysis software .
Cell migration assay
Cell migration was assessed using transwell permeable supports with an 8-μm pore membrane (Corning Costar, Tewksbury, MA, USA). C3H/10 T1/2 murine embryonic fibroblasts or RA-FLS were seeded in the upper chamber in DMEM supplemented with 1 % BSA. The lower chamber was filled with DMEM containing 0 %, 1 %, 6 %, or 10 % FBS. rhCTHRC1 (Sino Biological Inc., Beijing, China) or rhCTHRC1 (OriGene Technologies Inc., Rockville, MD, USA) were added to the upper chamber at 500–2,000 ng/ml. After 18 hours at 37 °C in 5 % CO2, cells were fixed with 0.3 % glutaraldehyde in PBS for 10 minutes at room temperature and then stained with toluidine blue. Upper surface of the membrane was cleaned to remove any non-migrated cells using a cotton swab. Membranes were peeled off, and mounted onto glass slides for microscopic examination. Transmigrated cells were counted at 200 × magnification in four to eight fields of view using bright field microscopy.
Murine NIH 3T3 fibroblasts were plated on collagen type I-coated μ-slides (IBIDI USA Inc., Madison, WI) and incubated for 4 hours at 37 °C in 5 % CO2/95 % air in complete DMEM media supplemented with 10 % FBS to insure complete cell adhesion on a bridge. Subsequently, rhCTHRC1 protein (Sino Biological Inc., Beijing, China) was added at 1000 ng/ml, and imaging has been performed for 14 hours in a serum-containing medium at 37 °C using time-lapse phase contrast microscopy with a Cell Observer microscope (Carl Zeiss Microscopy GmbH, Göttingen, Germany). Recorded stacks of images were transferred to ImageJ  and further analyzed with the MTrackJ plugin and Chemotaxis Tool (IBIDI USA Inc., Madison, WI, USA). The trajectories of individual cells were evaluated for accumulated distance migrated (Ad) and for cell velocity. Directness was calculated as a ratio of Euclidean distance (Ed) to the accumulated distance (Ed/Ad) (Fig. 6). Additionally, elongated morphology cell shape was studied (horizontal polarization), which was defined as maximal cell dimension using linear approximation. In preliminary experiments, we found that pro-motile and polarization effects of the CTHRC1 treatment were pronounced in the time window from 4–12 hours, and thereafter all calculations were performed for this time period.
Data were analyzed using Student’s t test for unpaired samples, using SPSS statistical software (SPSS Inc., Chicago, IL, USA). The Mann–Whitney U test for unpaired samples was performed using GraphPad Prism6 software (GraphPad Software Inc., La Jolla, CA, USA). P-values less than 0.05 were considered significant. Quantitative measurement of brown-colored DAB product of IHC staining was performed using ImageJ software  by color channel separation and densitometry of microscopic images at × 200–400 magnification. On each slide, at least five positively stained areas were analyzed for integrated brown-colored density; at least five slides per mouse (n = 5 per group) were analyzed.