Uridine Diphosphate Promotes Rheumatoid Arthritis Through P2Y6 Activation

BACKGROUND: Uridine diphosphate (UDP) is an extracellular nucleotide signaling molecule implicated in diverse biological processes via specific activation of pyrimidinergic receptor P2Y, G Protein-Coupled, 6 (P2Y6). There is very little knowledge about the function and mechanism of UDP in rheumatoid arthritis (RA). METHODS: This study used a quasi-targeted liquid chromatography-mass spectrometry (LC-MS) approach to investigate the unique expression of metabolites in RA synovial fluids (SF) (n = 10) with samples from osteoarthritis (OA) as controls (n = 10). RA fibroblast-like synoviocytes (FLSs) were collected from synovial tissues (n = 5) and cultured with UDP or MRS2578, a P2Y6 antagonist, and FLSs from OA were used as controls (n = 5). Rats with collagen-induced arthritis (CIA) were injected with UDP, MRS2578 or both (n = 9 for each group). P2Y6 expression was examined using real-time PCR, Western blotting and immunohistochemistry. Cell proliferation, apoptosis and migration of RA FLSs were measured using CCK-8 assay, real-time cell analysis, flow cytometry, wound healing assay and Transwell assay, respectively. The UDP levels in the culture medium, synovial fluid (n = 36) and peripheral blood (n = 36) of RA and CIA rats were measured using a Transcreener UDP Assay. Levels of proinflammatory cytokines were measured using a flow assay. Interleukin-6 (IL-6) levels were measured using ELISA and flow. RESULTS: LC-MS analysis detected significantly increased UDP levels in RA SF compared with OA SF, and the level was positively correlated with anticyclic citrullinated peptide (anti-CCP) and rheumatoid factor (RF)levels in RA. The increased UDP concentration was verified in the blood and synovial fluids of RA patients compared with samples from OA patients and healthy volunteers, respectively. UDP stimulated cell proliferation, migration and IL-6 secretion in RA FLSs and inhibited their apoptosis in culture, and MRS2578 inhibited these effects of UDP. UDP injection accelerated CIA and stimulated IL-6 production rather than other proinflammatory cytokines in the rat model, but simultaneous injection of MRS2578 suppressed these effects and alleviated CIA. P2Y6 expression was increased in RA and CIA synovial tissues. CONCLUSION: These results suggest that UDP is highly expressed in RA and stimulates RA pathogenesis by promoting P2Y6 activities to increase IL-6 production.


Evaluation of joint in ammation
Rats were sacri ced 20 days after the rst UDP injection. The in ammation curve was constructed according to the size of the hind paws, which was measured every other day using Vernier calipers. Bone erosion and cartilage destruction in the ankle joint and knee joint were assessed by X-ray imaging (75 kV, 195.3 mA) before sacri ce. The joint tissue within 0.5 cm of the knee joint of rats was collected, xed with 4% paraformaldehyde and embedded in para n. Hematoxylin-eosin staining was used to examine the pathological changes in joint tissues. According to clinical and histological evidence, the disease score is calculated as follows: 0 = normal joint; 1 = local swelling and/or erythema without histological damage; 2 = swelling and/or rigidity of the whole paw without histological damage; 3 = limb deformity with reversible histological damage; 4 = limb deformity accompanied by permanent histological damage such as bone or cartilage erosion.
Isolation and culture of human FLSs Synovial tissue samples from patients with RA (Patient No. 25 to No. 36, n = 12) or OA (Patient No. 25 to No. 36, n = 12) were minced into small pieces and digested for 4 h at 37 °C and 5% CO 2 in 3 mL of DMEM containing 4% type II collagenase (Solarbio, China) until the tissue pieces were dispersed into a cell suspension. The cell suspension was ltered through a 70 µm cell strainer and resuspended in DMEM containing 10% FBS. FLSs were incubated at 37 °C in a humidi ed incubator containing 5% CO 2 . Cells passed for 3-8 generations were used in subsequent experiments.

Measurement of UDP content
Peripheral blood samples from healthy donors and RA patients (Patient No. 1 to No. 36, n = 36) were collected into pyrogen-free and endotoxin-free test tubes with anticoagulants. SF samples from OA and RA patients (Patient No. 1 to No. 36, n = 36) were added to an equal volume of PBS. Rat SF and plasma were collected as described above. These samples were centrifuged at 1000 × g for 20 min at 4 °C, and the supernatant was carefully collected. The UDP content in the samples was measured using a Transcreener UDP Assay (BellBrook Labs, USA) via a uorescence polarization readout according to the manufacturer's protocol. A 15 µL mixture of reagents, including 8 nm UDP 2 antibody-Tb, 1 × Stop & Detect Buffer C and UDP HiLyte647 Tracer, was mixed with 5 µL of each sample in a 96-well plate. The plate was incubated for 1.5 h at room temperature and analyzed in a FlexStation® 3 Multimode Plate Reader (Molecular Devices, USA). The concentration of UDP was calculated by the standard curve prepared with standard UDP solution before analysis.
Measurement of cytokine concentrations in blood and culture medium RA FLSs (Patient No. 1 to No. 5, n = 5) were seeded in 96-well plates at a density of 3 × 10 4 cells per well and incubated overnight. UDP was dissolved in PBS (containing 0.1% DMSO). Cells were incubated with UDP at a nal concentration of 100 µM for 24 h. The supernatants were collected after centrifugation at quanti ed using a Human Th1/Th2 Subgroup Detection Kit (CellGene, China). In brief, antibodies speci c for IL-2, IL-4, IL-6, IL-10, TNF-α and IFN−γ were conjugated to uorescence-encoded beads, and beads with the biotinylated detection antibodies were mixed with the samples. Streptavidin-PE was added, and the mixture was incubated with shaking for 2 h at room temperature. The beads were washed and were then analyzed in a NovoCyte D2040R ow cytometer (ACEA Biosciences, USA). The data were analyzed using FlowJo software (Tree Star, USA).
Measurement of IL-6 levels using enzyme-linked immunosorbent assay (ELISA) RA FLSs and OA FLSs (Patient No. 1 to No. 5, n = 5) were treated with different concentrations (0 µM, 10 µM, 50 µM and 100 µM) of UDP (Sigma-Aldrich, Germany), and the supernatants were collected at 24 h. The concentration of IL-6 was measured with an ELISA kit (eBioscience, USA) according to the protocol. In brief, a 100 µL volume of the standard, control or sample was added to each well and incubated for 2 h at room temperature. After three washes, 200 µL of human IL-6 conjugate antibody was added to each well, incubated for 2 h at room temperature and washed three times. A 200 µL aliquot of substrate solution was then added to each well and incubated for 20 min at room temperature. Then, 50 µL of Stop Solution was added to each well, and the optical density of each well was measured at 450 nm in a microplate reader (BioTek, USA).
Measurement of IL-6 levels using ow assay RA FLSs (Patient No. 1 to No. 5, n = 5) were seeded in 96-well plates at a density of 3 × 10 4 cells per well and incubated overnight. UDP and MRS2578 were dissolved in PBS vehicle containing 0.1% DMSO. Cells were incubated with or without MRS2578 (Med Chem Express, USA) at a nal concentration of 10 µM for 1 h. UDP (Sigma-Aldrich, Germany) was then added at a nal concentration of 100 µM, and incubation was continued for 24 h. The supernatants were collected after centrifugation at 1000 × g for 20 min. The IL-6 concentrations in the supernatants were quanti ed using a human IL-6 ow assay kit (Cell Gene, China). In brief, anti-IL-6 antibodies were conjugated to uorescence-encoded beads, and the beads and biotinylated anti-IL-6 detection antibodies were mixed with the samples. Streptavidin-PE was added, and the mixture was incubated with shaking for 2 h at room temperature. The beads were washed and were then analyzed in a NovoCyte D2040R ow cytometer (ACEA Biosciences, USA). The data were analyzed using FlowJo software (Tree Star, USA).
Rat plasma was collected from the inferior vena cava, and the IL-6 level was measured using a similar protocol with the rat IL-6 capture bead B6 product commercially obtained from BioLegend. The data were analyzed using LEGENDplex v8.0 software (BioLegend). Evaluation of FLS proliferation by a CCK-8 assay RA FLSs and OA FLSs (Patient. 1 to No. 5, n = 5) were treated with different concentrations (0 µM, 10 µM, 50 µM or 100 µM) of UDP for 0, 6, 12 and 24 h. A 10 µL volume of CCK-8 solution (Dojindo, Japan) was added to each well and incubated for an additional 4 h. The absorbance was measured at 450 nm in a spectrophotometer (BioTek, USA).

Evaluation of FLS proliferation using real-time cell analysis (RTCA)
A dual-plate RTCA instrument (ACEA Biosciences, USA) (Patient No. 6 to No. 10 n = 5) was placed in a humidi ed incubator maintained at 37 °C and 5% CO 2 . RA FLSs were seeded in cell culture E-plates (1 × 10 4 cells per well) (ACEA Biosciences, USA) and treated with 100 µM UDP with or without MRS2578 at a nal concentration of 10 µM for 3 days. The 96-well E-plate was monitored every 30 min for 48 h, and cell proliferation was monitored in real time by measuring the electrical impedance using the xCELLigence RTCA TP System (ACEA Biosciences, USA). The cell growth curves were automatically recorded based on continuous quantitative monitoring of cell proliferation. The data were analyzed with Real-Time Cell Analyzer software (version 1.2).
Detection of FLS apoptosis via ow cytometry RA FLSs and OA FLSs (Patient No. 6 to No. 10, n = 5) were treated with or without MRS2578 (MedChemExpress, USA) at a nal concentration of 10 µM for 1 h. Then, UDP at a nal concentration of 100 µM was added, and incubation was continued for 24 h. Cells (6 × 10 4 ) were then collected and resuspended in binding buffer. An Annexin V-FITC-conjugated antibody and a PI-conjugated antibody (BioLegend) were then added to the suspended cells. Apoptosis was detected by ow cytometry.
Cell migration assay RA FLSs and OA FLSs (Patient No. 6 to No. 10, n = 5) were seeded in 6-well plates. When the cells were 80-90% con uent, the wound healing assay was conducted by scratching the cell layer in each well with a sterile P200 pipette tip. The cells were preincubated with or without MRS2578 (MedChemExpress, USA) at a nal concentration of 10 µM for 1 h. UDP at a nal concentration of 100 µM was then added, and incubation was continued for 24 h. The cells were photographed at 0 h and 24 h (Olympus IX51, Japan), and the wound area was calculated with ImageJ software (NIH, Bethesda, MD, USA).
Transwell assay RA FLSs and OA FLSs (Patient No. 6 to No. 10, n = 5) (1 × 10 4 cells/mL) in serum-free medium were seeded in the upper compartments of Matrigel Invasion Chambers (Corning, USA). Medium containing 10% FBS was added to the lower compartments of the chambers. The cells were incubated with or without MRS2578 (MedChemExpress, USA) at a nal concentration of 10 µM for 1 h. UDP (Sigma-Aldrich, Germany) at a nal concentration of 100 µM was then added, and incubation was continued for 24 h.
The cells on the top surface of the membrane were removed with cotton swabs, and cells that penetrated to the bottom surface of the membrane were stained with crystal violet. Images were acquired by uorescence microscopy (Olympus IX51, Japan), and the cells were quanti ed with ImageJ software (NIH, Bethesda, MD, USA).

Sources of microarray data
The expression level of P2Y6 in RA and OA synovial tissue was analyzed in four published gene expression pro le datasets (dataset type: expression pro ling by array) in the Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geopro les) database. The expression data of 10 patients with RA and 6 OA controls from dataset GDS5402/208373_s_at, 5 patients with RA and 5 OA controls from dataset GDS2126/38222_at, 10 patients with RA and 10 OA controls from dataset GDS5401/208373_s_at, and 13 patients with RA and 10 OA controls from dataset GDS5403/208373_s_at were analyzed in SPSS software v. 21.0 (IBM, USA) using an unpaired Student's ttest.
Examination of P2Y6 expression using immunohistochemical staining Para n sections of human synovial tissues (Patient No. 20 to No. 24, n = 5) were incubated rst with a rabbit anti-P2Y6 antibody (1:200, Abcam, ab198805) at 4 °C overnight and then with HRP-conjugated goat anti-rabbit IgG (ab205718). Sections were then treated with diaminobenzidine (DAB) and counterstained with hematoxylin. The results were analyzed and expression was quanti ed in ImageJ software (NIH, Bethesda, MD, USA).

Statistical analysis
Statistical analyses were performed using GraphPad Prism 7.0 (GraphPad, USA) and SPSS software v.21.0 (IBM, USA). The signi cance of differences between groups was evaluated using Student's unpaired t-test. Differences with p values of < 0.05 were considered signi cant.

The effect of UDP on RA FLSs
Cultured RA FLSs (n = 5) and OA FLSs (n = 5) were treated with different concentrations of UDP (0 µM, 10 µM, 50 µM and 100 µM) for different times (0 h, 6 h, 12 h and 24 h). The CCK-8 assay showed increased proliferation of RA FLSs in the presence of 10 µM, 50 µM and 100 µM UDP (p = 0.002, 0.0003, and 0.0002, respectively) compared with that in the PBS-treated control group (Fig. 3A). However, the CCK-8 assay did not show a signi cant change in the proliferation of OA FLSs in the presence of 10 µM, 50 µM and 100 µM UDP (p = 0.96, 0.90, and 0.81, respectively) compared with that in the PBS-treated control group (Fig. 3B). The Annexin V/PI apoptosis analysis showed that the apoptosis rate of RA FLSs was decreased in the presence of UDP (100 µM) compared with that in the PBS-treated control group (p = 0.0316) (Fig. 3C). However, the Annexin V/PI apoptosis analysis detected little change in OA FLS apoptosis in the presence of UDP (p = 0.3317) compared with that in the PBS-treated control culture (Fig. 3D).
We used wound healing and Transwell assays to evaluate the effect of UDP on the migration of RA FLSs. The wound healing assays showed that RA FLS migration was increased in the presence of UDP (100 µM) (p = 0.0022) compared with that of control FLSs treated with PBS (Fig. 3E). The Transwell assays also showed that RA FLS migration was increased in the presence of UDP (100 µM) (p = 0.004) (Fig. 3F) compared with that of PBS-treated control FLSs. However, the wound healing assay showed only a slight change in OA FLS migration in the presence of UDP (100 µM) (p = 0.59) compared with that of control FLSs treated with PBS (Fig. 3G). The Transwell assay also showed only a slight change in OA FLS migration in the presence of UDP (100 µM) (p = 0.32) compared with that of FLSs treated with PBS (Fig. 3H).
We used ow cytometry to examine proin ammatory cytokine production in the FLS cultures. The assay showed signi cantly elevated IL-6 levels in the culture medium of RA FLSs in the presence of 100 µM UDP (2203 ± 712.2, p = 0.044), but the concentrations of IL-2, IL-4, IL-10, TNF-α and IFN-γ were not signi cantly changed ( (Fig. 4B).
An IL-6 ELISA was performed to verify the above results. This assay also showed signi cantly increased IL-6 levels in the culture medium of RA FLSs in the presence of 10 µM, 50 µM and 100 µM UDP (p = 0.022, 0.0205, and 0.0003, respectively) compared with that in samples from cells treated with PBS (Fig. 4C). However, ELISA showed only slight changes in IL-6 levels in the culture medium of OA FLSs in the presence of 10 µM, 50 µM and 100 µM UDP (p = 0.559, 0.088, and 0.010, respectively) (Fig. 4D). The above results indicated that UDP activated RA FLSs and induced IL-6 secretion but did not similarly affect OA FLSs.

Regulation of P2Y6 expression on the effects of UDP
The abundance of P2Y6 mRNA in RA and OA synovial tissue was analyzed in datasets from the GEO database (dataset type: expression pro ling by array). The expression data (expression values) of 10 patients with RA and 6 OA controls from dataset GDS5402/208373_s_at, 5 patients with RA and 5 OA controls from dataset GDS2126/38222_at, 10 patients with RA and 10 OA controls from dataset GDS5401/208373_s_at, and 13 patients with RA and 10 OA controls from dataset GDS5403/208373_s_at were analyzed in SPSS software v.21.0. Signi cantly increased transcription of P2Y6 was found in RA synovial tissues (259.6 ± 24.53, n = 38) compared with OA samples (171.3 ± 18.85, n = 31) (Student's unpaired t-test, p = 0.0076) (Fig. 5A).
To verify the effect of UDP on RA FLSs, RA FLSs (n = 5) were cultured with both UDP (100 µM) and MRS2578 (10 µM), the chemical inhibitor of P2Y6. Cell proliferation was investigated using RTCA, apoptosis was detected with an annexin V/PI apoptosis assay, and IL-6 secretion was measured using a ow cytometric bead assay. Compared with that of RA FLSs cultured with PBS, RA FLS proliferation was increased in the presence of UDP (p = 0.0007) and was decreased in the presence of MRS2578 alone (p = 0.0017) and of UDP and MRS2578 together (p = 0.0024). The above results demonstrated that RA FLS proliferation was decreased in the presence of MRS2578 alone (p < 0.0001) and of UDP and MRS2578 together (p < 0.0001) (Fig. 6A). Compared with that in the PBS-treated culture, RA FLS apoptosis was decreased in the presence of UDP alone (p = 0.0440), increased in the presence of MRS2578 alone (p = 0.0285), and unchanged in the presence of UDP and MRS2578 together (p = 0.0917). The above results demonstrated that RA FLS apoptosis was increased in the presence of MRS2578 alone (p = 0.0339) and of UDP and MRS2578 together (p = 0.0476) (Fig. 6B). In addition, compared with that in the PBS-treated culture, IL-6 secretion in RA FLSs was increased in the presence of UDP (p = 0.0051) and was decreased in the presence of MRS2578 alone (p = 0.0021) and of UDP and MRS2578 together (p = 0.0023) (Fig. 6C). The wound healing assay showed that compared with that in the PBS-treated control culture, RA FLS migration was increased in the presence of UDP (p = 0.0016) and was decreased in the presence of MRS2578 alone (p = 0.0094) and of UDP and MRS2578 together (p = 0.0007). The above results demonstrated that RA FLS migration was decreased in the presence of MRS2578 alone (p < 0.0001) and of UDP and MRS2578 together (p = 0.0031) (Fig. 6D). Moreover, the Transwell assay showed that compared with that in the PBS-treated control culture, RA FLS migration was increased in the presence of UDP (p = 0.0003) and was decreased in the presence of MRS2578 (p = 0.0088) and of UDP and MRS2578 together (p = 0.0328). The above results demonstrated that RA FLS migration was decreased in the presence of MRS2578 alone (p = 0.0014) and of UDP and MRS2578 together (p = 0.0063) (Fig. 6E).
The effect of UDP on CIA Rats induced with collagen II were simultaneously injected with UDP, MRS2578 or both UDP and MRS2578. The disease score as assessed by toe swelling was increased, and radiological signs (soft tissue swelling, new bone formation and marginal osseointegration) and histochemical staining were signi cantly enhanced in CIA rats compared with normal control rats (p < 0.0001), indicating successful establishment of CIA in the rats. The disease score was signi cantly increased in CIA rats treated with UDP (p = 0.0052) but decreased in CIA rats treated with MRS2578 (p = 0.0193) or both UDP and MRS2578 (p = 0.0331) compared with CIA rats. In addition, compared with that in CIA rats treated with UDP, the disease score was decreased in CIA rats treated with MRS2578 (p = 0.0002) or with both UDP and MRS2578 (p = 0.0002). The disease score did not differ signi cantly between CIA rats treated with MRS2578 alone and CIA rats treated with both UDP and MRS2578 (p = 0.7332) (Fig. 7A, B). Compared with that in normal control rats, paw in ammation in CIA rats was signi cantly exacerbated, indicating successful establishment of CIA in the rats. Paw in ammation was signi cantly exacerbated in CIA rats treated with UDP (p = 0.0058) but alleviated in CIA rats treated with MRS2578 (p = 0.024) or with both UDP and MRS2578 (p = 0.004) compared with CIA rats. Compared with that in CIA rats treated with UDP, paw in ammation was alleviated in CIA rats treated with MRS2578 (p < 0.0001) or with both UDP and MRS2578 (p < 0.0001). Paw in ammation did not differ signi cantly between CIA rats treated with MRS2578 alone and CIA rats treated with both UDP and MRS2578 (p = 0.51) (Fig. 7C).
SF was also collected from rats on day 20 after the rst UDP injection, and the cytokine concentrations were measured by ow cytometry. Compared with the corresponding concentrations in the normal control group, the concentrations of IL-6, GM-CSF and TNF-α in the CIA control group were signi cantly increased (p = 0.0299, 0.0250, and 0.0376, respectively), the concentrations of IL-10 were signi cantly decreased (p = 0.0317), and the concentrations of IL-2, IL-4, IL-5, IL-13, and IFN-γ were not signi cantly changed. Compared with that in CIA rats, the IL-6 level was signi cantly increased in CIA rats treated with UDP (p = 0.0053) but decreased in CIA rats treated with MRS2578 (p = 0.0040) or with both UDP and MRS2578 (p = 0.0114). Compared with that in CIA rats treated with UDP, the IL-6 level was decreased in CIA rats treated with MRS2578 (p = 0.0002) or with both UDP and MRS2578 (p = 0.0004). However, the IL-6 level did not differ signi cantly between CIA rats treated with MRS2578 alone and CIA rats treated with both UDP and MRS2578 (p = 0.2901). Moreover, the concentrations of IL-2, IL-4, IL-5, IL-10, IL-13, GM-CSF, IFN-γ and TNF-α did not differ signi cantly (Fig. 7D). Cytokine expression in the SF of rats was also evaluated by IL-6 ELISA. The IL-6 level was signi cantly increased in CIA rats treated with UDP (p = 0.0486) but decreased in CIA rats treated with MRS2578 (p = 0.0467) or with both UDP and MRS2578 (p = 0.293) compared with CIA rats. Compared with that in CIA rats treated with UDP, the IL-6 level was decreased in CIA rats treated with MRS2578 (p = 0.0005) or with both UDP and MRS2578 (p = 0.0002). However, the IL-6 level did not differ signi cantly between CIA rats treated with MRS2578 alone and CIA rats treated with both UDP and MRS2578 (p = 0.908) (Fig. 7E).
We examined UDP levels in the peripheral blood and SF of CIA rats using the uorescence polarization method. The UDP level in peripheral blood was signi cantly higher in CIA rats than in normal control rats (p = 0.0163). Compared with that in CIA rats, the peripheral blood UDP level was signi cantly increased in CIA rats treated with UDP (p = 0.0482) or both UDP and MRS2578 (p = 0.0362) but was not signi cantly different in CIA rats treated with MRS2578 (p = 0.3711). Compared with that in CIA rats treated with UDP, the peripheral blood UDP level was decreased in CIA rats treated with MRS2578 (p = 0.0312) but was not signi cantly different in CIA rats treated with both UDP and MRS2578 (p = 0.9421). The UDP level in peripheral blood was signi cantly higher in CIA rats treated with both UDP and MRS2578 than in CIA rats treated with MRS2578 (p = 0.0231) (Fig. 8A). The UDP level in SF was signi cantly higher in CIA rats than in normal control rats (p = 0.0090). Compared with that in CIA rats, the UDP level in SF was signi cantly increased in CIA rats treated with UDP (p = 0.0066) or both UDP and MRS2578 (p = 0.0074) but was not signi cantly different in CIA rats treated with MRS2578 (p = 0.4286). Compared with that in CIA rats treated with UDP, the UDP level in SF was decreased in CIA rats treated with MRS2578 (p = 0.0022) but was not signi cantly different in CIA rats treated with both UDP and MRS2578 (p = 0.5821). Finally, the UDP level in SF was signi cantly higher in CIA rats treated with both UDP and MRS2578 than in CIA rats treated with MRS2578 alone (p = 0.0019) (Fig. 8B).
We also examined P2Y6 expression in synovial tissues of the model rats by real-time PCR and Western blot analysis. P2Y6 mRNA expression in synovial samples was signi cantly higher in CIA rats than in normal control rats (p = 0.0017). P2Y6 mRNA expression in synovial tissues did not differ signi cantly between CIA rats and CIA rats treated with UDP alone (p = 0.196), MRS2578 alone (p = 0.211) or UDP and MRS2578 together (p = 0.721). P2Y6 mRNA expression in synovial tissues did not differ signi cantly between CIA rats treated with UDP and CIA rats treated with MRS2578 alone (p = 0.0581) or with UDP and MRS2578 together (p = 0.1091). P2Y6 mRNA expression in synovial tissues did not differ signi cantly between CIA rats treated with MRS2578 alone and CIA rats treated with UDP and MRS2578 together (p = 0.348) (Fig. 8C). However, P2Y6 protein expression in synovial tissues was signi cantly higher in CIA rats than in normal control rats (p = 0.0049). However, P2Y6 protein expression in synovial tissues did not differ signi cantly between CIA rats and CIA rats treated with UDP alone (p = 0.476), MRS2578 alone (p = 0.308) or UDP and MRS2578 together (p = 0.545). Moreover, P2Y6 protein expression in synovial tissues did not differ signi cantly between CIA rats treated with UDP and CIA rats treated with MRS2578 alone (p = 0.128) or with UDP and MRS2578 together (p = 0.267). P2Y6 protein expression in synovial tissues also did not differ signi cantly between CIA rats treated with MRS2578 alone and CIA rats treated with UDP and MRS2578 together (p = 0.765) (Fig. 8D).

Discussion
In this study, via metabolomic analysis, we found signi cantly increased UDP levels in RA SF compared with OA SF. We con rmed this nding in blood and SF by comparing samples from 36 RA patients and 36 OA patients using the Transcreener UDP Assay. Furthermore, we found increased levels of UDP in blood and SF from CIA rats compared with normal control rats. These results suggest a high level of UDP in RA and CIA. Furthermore, the UDP level was moderately correlated with the levels of anti-CCP and RF in RA SF, indicating the potential role of UDP in RA.
We continued by investigating the effect of a high UDP level on RA. UDP injection signi cantly aggravated paw in ammation in CIA rats. Additionally, UDP stimulated the proliferation and migration of RA FLSs in vitro and suppressed their apoptosis, indicating the activating effects of UDP on RA FLSs. UDP also increased IL-6 secretion by cultured RA FLSs and in CIA rats but did not affect the production of other cytokines, such as IL-2, IL-4, IL-10, TNF-α and IFN-γ. IL-6 plays a key role in local and systemic manifestations of RA [19]. Blockade of IL-6 has been suggested to be an effective method for RA treatment [20,21]. The above results suggest that a high UDP level stimulates the pathogenic progression of RA. This study is the rst to report the effects of a high UDP level on RA, although many studies have reported that UDP activates in ammatory responses such as phagocytosis and cytokine/chemokine production [17,22,23].
UDP plays a role via P2Y6. The human P2Y6 receptor (hP2Y6) is a member of the G protein-coupled pyrimidinergic P2 receptor family that responds speci cally to the extracellular nucleotide UDP. P2Y6 is expressed in neutrophils, macrophages, dendritic cells, eosinophils, B cells and T cells and plays roles in apoptosis and cell differentiation, maturation and migration [24]. In our study, we found high P2Y6 expression in RA synovial tissues using real-time PCR, Western blotting and immunohistochemistry. We also found increased expression of P2Y6 in CIA synovial tissues. When RA FLSs were cultured with both UDP and MRS2578, a P2Y6 antagonist, their proliferation and IL-6 secretion were signi cantly suppressed, and the apoptosis rate was increased. CIA rats injected with MRS2578 or with both UDP and MRS2578 showed decreased paw in ammation and IL-6 production. UDP had little effect on OA FLS proliferation and IL-6 secretion, and P2Y6 expression was relatively low in OA synovial tissues. These observations suggest that UDP plays a stimulatory role in RA via P2Y6. Increased UDP levels and high P2Y6 expression stimulate RA and CIA progression. Targeting P2Y6 receptors might be useful for the treatment of RA. However, UDP did not change P2Y6 expression; P2Y6 protein expression in synovial tissues was not signi cantly changed in CIA rats treated with UDP alone, MRS2578 alone or UDP and MRS2578 together. The reason that P2Y6 expression is elevated in RA and CIA is unknown. However, high levels of UDP and P2Y6 cooperatively promoted RA pathogenesis.
Extracellular nucleotides (ATP, ADP, UTP and UDP) exert proin ammatory effects through the activation of P2 purinergic receptors such as P2 × 7 and P2Y6 [4,17,25]. ATP can act as a danger signal that can cause systemic in ammatory response syndrome [25,26]. Accumulating evidence has suggested that P2 × 7, a receptor for ATP, is a critical regulator and potential target of RA [27,28]. UDP is an important extracellular nucleotide signaling molecule implicated in diverse biological processes via speci c activation of metabotropic pyrimidine and purine nucleotide receptors (P2Y receptors). Pyrimidine and purine metabolism are components of nucleotide metabolism.
Purine metabolism is activated in RA relative to OA [29]. Methotrexate, a rst-line drug for RA, can alleviate RA progression by inhibiting pyrimidine metabolism and purine metabolism [30,31].
Le unomide, a selective inhibitor of de novo pyrimidine synthesis that alters pyrimidine metabolism, has been successfully used to treat RA and psoriatic arthritis for many years [32,33]. Lapachol, a compound targeting pyrimidine metabolism, can ameliorate experimental autoimmune arthritis [34]. These results support our nding indicating the important role of UDP-related nucleotide metabolism in RA.
MRS2578 was found to inhibit the release of IL-6 and IL-8/keratinocyte chemoattractant (IL-8/KC) by lung epithelial cells in vivo, whereas intrapulmonary application of the P2Y6 receptor agonist UDP increased the bronchoalveolar levels of IL-6 and KC. In addition, selective activation of P2Y6 receptors was found to induce the secretion of IL-6 and KC/IL-8 by murine and human lung epithelial cells in vitro [35]. Application of pressure was found to induce IL-6 expression through the P2Y6 receptor in human dental pulp cells [36]. Treatment with monosodium urate crystals stimulated normal human keratinocytes to produce IL-1α, IL-8/CXCL8, and IL-6 through P2Y6 receptors. In addition, treatment with either P2Y6speci c antagonists or P2Y6 antisense oligonucleotides signi cantly inhibited the production of IL-1α, IL-8/CXCL8 and IL-6 by human keratinocytes [37]. Activation of the P2Y6 receptor by its natural ligand, UDP, or its speci c agonist, MRS2693, led to the production of IL-6 and IL-8 [38]. In vitro studies demonstrated that proliferation and IL-6 secretion are P2Y6 receptor-mediated processes in lung broblast cells [39]. MRS2578 treatment was found to diminish chronic constriction injury-induced increases in P2Y6 mRNA expression and IL-6 secretion as well as JAK2/STAT3 mRNA expression and phosphorylation modi cation in spinal cord tissues [40]. UDP was also found to induce the production of the proin ammatory chemokines monocyte chemotactic protein-1 (MCP-1) and Interferon-γ (IFN-γ)-induced protein 10 (IP-10 or CXCL-10) in hP2Y6-transfected promonocytic U937 cells but not in 1321N1 astrocytoma cell lines stably transfected with hP2Y6. Moreover, UDP was found to induce the production of IL-8 but not TNF-alpha in human 1321N1 astrocytoma cells stably transfected with hP2Y6. Therefore, the immunostimulatory effect of UDP/P2Y6/IL-6 signaling on the production of proin ammatory cytokines is selective and cell type-dependent [22]. The above observations support our ndings regarding the stimulation of the UDP/P2Y6/IL-6 pathway during RA progression.
In summary, this study showed that the level of UDP is increased in RA and CIA and that UDP stimulates proliferation, cell migration and IL-6 secretion in cultured RA FLSs and CIA rats. Additionally, P2Y6 expression was found to be increased in RA and CIA synovial tissues. Treatment with the P2Y6 antagonist MRS2578 inhibited the effects of UDP on proliferation and IL-6 secretion in RA FLSs and CIA rats. These results suggest that UDP is highly expressed in RA and stimulates RA pathogenesis by promoting P2Y6 activities to increase IL-6 production.      Effect of MRS2578 on proliferation and IL-6 levels in cultured RA FLSs. Cultured RA FLSs were treated with UDP (100 μM) and/or MRS2578 (10 μM). (A) Cell proliferation was assessed using RTCA and statistical analysis. (B) Apoptosis was assessed using ow cytometry and statistical analysis. (C) IL-6 secretion was assessed using a ow cytometric bead assay and statistical analysis. (D) The migration of RA FLSs was assessed using a wound healing assay and statistical analysis. (E) RA FLS migration was assessed using a Transwell assay and statistical analysis. *p < 0.05, **p < 0.01; ***p < 0.001.

Figure 7
Effect of the P2Y6 inhibitor on CIA rats treated with UDP. (A) X-ray images and histochemical staining images of joint in ammation in CIA rats treated with UDP and/or MRS2578. (B) Disease scores were quanti ed based on histologic evidence. (C) In ammation curve analysis based on paw in ammation.
(D) Cytokine release into rat PB was assessed using a ow cytometric bead assay. (E) Cytokine release in rat SF using a ow cytometric bead assay. (F) IL-6 release into rat SF was assessed using ELISA. *p<0.05, **p<0.01, and ***p<0.001.