Immune complexes containing citrullinated fibrinogen are present in the sera and synovium of rheumatoid arthritis patients and potentially contribute to synovitis. However, fibrinogen can inhibit the osteoclastogenesis of precursor cells. We investigated the direct effect of citrullinated fibrinogen on osteoclastogenesis to understand the role of citrullination on bone erosion of rheumatoid arthritis patients. We evaluated the fibrinogen citrullination sites using mass spectrometry and quantified osteoclast-related protein and gene expression levels by Western blotting, microarray, and real-time polymerase chain reaction. Differences in spectral peaks were noted between fibrinogen and citrullinated fibrinogen at five sites in α-chains, two sites in β-chains, and one site in a γ-chain. Transcriptome changes induced by fibrinogen and citrullinated fibrinogen were identified and differentially expressed genes grouped into three distinctive modules. Fibrinogen was then citrullinated in vitro using peptidylarginine deiminase. When increasing doses of soluble fibrinogen and citrullinated fibrinogen were applied to human CD14+ monocytes, citrullination restored osteoclastogenesis-associated changes, including NF-ATc1 and ß3-integrin. Finally, citrullination rescued the number of osteoclasts by restoring fibrinogen-induced suppression of osteoclastogenesis. Taken together, the results indicate that the inhibitory function of fibrinogen on osteoclastogenesis is reversed by citrullination and suggest that citrullinated fibrinogen may contribute to erosive bone destruction in rheumatoid arthritis.

Introduction: C-X-C motif chemokine 10 (CXCL10) is a chemokine that plays a critical role in the infiltration of T cells in autoimmune diseases and is reported to be expressed in muscle tissue of polymyositis. To determine the therapeutic efficacy of CXCL10 blockade, we investigated the role of CXCL10 and the effect of anti-CXCL10 antibody treatment in C protein-induced myositis (CIM), an animal model of polymyositis.Methods: CIM was induced with human skeletal muscle C protein fragment in female C57BL/6 mice. Immunohistochemistry of CXCL10 and C-X-C motif chemokine receptor 3 (CXCR3) and measurement of serum CXCL10 were performed. Cell surface markers and interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) in CIM lymph node cells was investigated by flow cytometry. Mice with CIM were treated with anti-CXCL10 antibody or control antibody (anti-RVG1) and the inflammation in muscle tissue was assessed.Results: Immunohistochemistry showed increased expression of CXCL10 and CXCR3 in the inflammatory lesions of muscle in CIM. Especially, CD8+ T cells invading myofiber expressed CXCR3. Serum level of CXCL10 was increased in CIM compared to the level in normal mice (normal mouse, 14.3 ± 5.3 pg/ml vs. CIM, 368.5 ± 135.6 pg/ml,P < 0.001). CXCR3 positivity in CD8+ T cells was increased compared to that of CD4+ T cells in the lymph node cells of CIM (CXCR3+ among CD8+ T cell, 65.9 ± 2.1% vs. CXCR3+ among CD4+ T cell, 23.5 ± 4.7%,P<0.001). Moreover, IFN-γ+ cells were increased among CXCR3+CD8+ T cells compared to CXCR3–CD8+ T cells (CXCR3+CD8+ T cell, 28.0 ± 4.2% vs. CXCR3-CD8+ T cell, 9.5 ± 1.5%,P = 0.016). Migration of lymph node cells was increased in response to CXCL10 (chemotactic index was 1.91 ± 0.45). CIM mice treated with anti-CXCL10 antibody showed a lower inflammation score in muscles than those with anti-RVG1 (median, anti-CXCL10 treatment group, 0.625 vs. anti-RVG1 treatment group, 1.25,P = 0.007).Conclusions: CXCL10/CXCR3 expression was increased in the inflammation of CIM model and its blockade suppressed inflammation in muscle.

Introduction: Statins, hydroxymethylglutaryl-coenzyme A reductase inhibitors, have been reported to have antiinflammatory and/or immunomodulatory effects and prophylactic and therapeutic effects in collagen-induced arthritis, an experimental model of rheumatoid arthritis (RA). The authors undertook to determine the effect of atorvastatin on the expressions of osteoprotegerin (OPG) and receptor activator of nuclear factor κB ligand (RANKL) in RA fibroblast-like synoviocytes (FLSs), to identify the mechanisms responsible for these effects, and to determine whether the statin inhibits osteoclastogenesis. Methods: FLSs isolated from five RA patients were cultured in the presence of 20 ng/ml of tumor necrosis factor-α (TNF-α) with or without atorvastatin. RANKL expressions were assayed with Western blotting and enzyme-linked immunosorbent assay. RANKL, RANK, and OPG expression were assayed with reverse transcription-polymerase chain reaction (RT-PCR). Osteoclast formation was assayed by counting cells after staining for tartrate-resistant acid phosphatase in cocultures of peripheral blood mononuclear cells (PBMCs) and RA FLSs. Results: Atorvastatin inhibited the expression of RANKL in RA FLSs in a dose-dependent manner, and the suppression of RANKL was prevented by mevalonate. However, OPG expression was not affected by atorvastatin in RA FLSs, and atorvastatin did not affect RANK expression in CD14⁺ cells. Conversely, atorvastatin suppressed TNF-α-induced p38 phosphorylation in RA FLSs and significantly reduced TRAP-positive multinucleated osteoclast formation in the coculture of PBMCs and RA FLSs. Conclusion: These results suggest that atorvastatin inhibits osteoclastogenesis and bone destruction in RA patients.

Behçet’s disease (BD) susceptibility had been associated with single-nucleotide polymorphisms (SNPs) in IL23R–IL12RB2, IL10, STAT4, or ERAP1 locus in Japanese, Turkish, Chinese, and other populations, but not in a Korean genome-wide association study (GWAS). We aimed to fine-map BD risk association of these four loci using extensive imputation and additional genotyping for replication. In the discovery phase, 369 patients with BD enrolled in the previous Korean GWAS and 2000 controls retrieved from a population-based cohort of healthy Koreans were imputed for their genotypes of all SNPs in the four loci using the Asian data of the 1000 Genomes Project as reference. For genotype imputation of ERAP1 SNPs, the adjacent ERAP2 SNPs were also covered. For the 10 most significantly associated SNPs (8 imputed and 2 GWAS-genotyped), an additional 84 patients with BD and 283 healthy controls were genotyped for replication. The results from the discovery and replication phases were pooled for meta-analysis using the Mantel-Haenszel test to estimate the odds ratio (OR) and 95% confidence interval (CI). An IL23R–IL12RB2 intergenic SNP rs1495965 was significantly associated with BD risk (OR (95% CI) = 1.5 (1.3, 1.7), P = 2.5 × 10−7) in the pooled meta-analysis of the discovery (1.4 (1.2, 1.7), P = 4.9 × 10−7) and replication (1.9 (1.3, 2.6), P = 6.0 × 10−4) phases. BD risk association was fine-mapped on the intergenic region rather than the two flanking genes, as rs1495966 and rs4655535, almost perfectly correlated with rs1495965 (r 2 = 0.99), were also located in the same intergenic region. Consistent with previous reports, the P values tended to be lower within IL23R than IL12RB2. On the other hand, several IL10 SNPs were suggested for association in the discovery phase but all failed in the replication phase. No SNP in ERAP1–ERAP2 and STAT4 was suggested even in the discovery phase. BD susceptibility association was fine-mapped on the intergenic region between IL23R and IL12RB2 as marked by three correlated SNPs, rs1495965, rs1495966, and rs4655535.