Masaki Katayama

Anti-citrullinated peptide antibody-negative RA is a genetically distinct subset: a definitive study using only bone-erosive ACPA-negative rheumatoid arthritis

Objectives. ACPA is a highly specific marker for RA. It was recently reported that ACPA can be used to classify RA into two disease subsets, ACPA-positive and ACPA-negative RA. ACPA-positive RA was found to be associated with the HLA-DR shared epitope (SE), but ACPA negative was not. However, the suspicion remained that this result was caused by the ACPA-negative RA subset containing patients with non-RA diseases. We examined whether this is the case even when possible non-RA ACPA-negative RA patients were excluded by selecting only patients with bone erosion. Methods. We genotyped HLA-DRB1 alleles for 574 ACPA-positive RA, 185 ACPA-negative RA (including 97 erosive RA) and 1508 healthy donors. We also tested whether HLA-DR SE is associated with RF-negative or ANA-negative RA. Results. ACPA-negative RA with apparent bone erosion was not associated with SE, supporting the idea that ACPA-negative RA is genetically distinct from ACPA-positive RA. We also tested whether these subsets are based on autoantibody-producing activity. In accordance with the ACPA-negative RA subset, the RF-negative RA subset showed a clearly distinct pattern of association with SE from the RF-positive RA. In contrast, ANA-negative as well as ANA-positive RA was similarly associated with SE, suggesting that the subsets distinguished by ACPA are not based simply on differences in autoantibody production. Conclusions. ACPA-negative erosive RA is genetically distinct from ACPA-positive RA.

Neutrophils Are Essential As A Source Of Il-17 In The Effector Phase Of Arthritis

Objective Th17 has been shown to have a pivotal role in the development of arthritis. However, the role of IL-17 in the T cell-independent effector phase has not fully been examined. We investigated whether IL-17 is involved in the effector phase of arthritis by using K/BxN serum-induced arthritis model. Methods K/BxN serum was transferred into IL-17 knockout (KO) mice, SCID mice and their control mice, and arthritis was evaluated over time. In order to clarify the source of IL-17 in the effector phase, neutrophils or CD4+ T cells collected from IL-17 KO or control mice were injected into IL-17 KO recipient mice together with K/BxN serum. To examine if neutrophils secrete IL-17 upon stimulation, neutrophils were stimulated with immune complex in vitro and IL-17 in the supernatant was measured by ELISA. Results K/BxN serum-induced arthritis was much less severe in IL-17 KO mice than in WT mice. Since K/BxN serum-transferred SCID mice developed severe arthritis with high serum IL-17 concentration, we speculated neutrophils are the responsible player as an IL-17 source. When wild type (WT) but not IL-17 KO neutrophils were co-injected with K/BxN serum into IL-17 KO mice, arthritis was exacerbated, whereas co-injection of WT CD4+ T cells had no effect. In vitro, stimulation of neutrophils with immune complexcaused IL-17 secretion. Conclusions Neutrophils are essential as a source of IL-17 in the effector phase of arthritis. The trigger of secreting IL-17 from neutrophils may be immune complex.

A large-scale association study identified multiple HLA-DRB1 alleles associated with ACPA-negative rheumatoid arthritis in Japanese subjects

Background HLA-DRB1 is associated with rheumatoid arthritis (RA). However, it has recently been suggested that HLA-DRB1 is only associated with patients with RA who have anticitrullinated peptide/protein antibodies (ACPA), which are specific to RA. Objective To elucidate whether specific HLA-DR alleles are associated with ACPA-negative RA development. Methods HLA-DRB1 typing was carried out in 368 Japanese ACPA-negative patients with RA and 1508 healthy volunteers as the first set, followed by HLA-DRB1 typing of 501 cases and 500 controls as the second set. The HLA-DRB1 allele frequency and diplotype frequency were compared in each group, and the results of the two studies were combined to detect HLA-DRB1 alleles or diplotypes associated with ACPA-negative RA. Results HLA-DRB1*12:01 was identified as a novel susceptibility allele for ACPA-negative RA (p=0.000088, OR=1.72, 95% CI 1.31 to 2.26). HLA-DRB1*04:05 and *14:03 showed moderate associations with ACPA-negative RA (p=0.0063, OR=1.26, 95% CI 1.07 to 1.49 and p=0.0043, OR=1.81, 95% CI 1.20 to 2.73, respectively). The shared epitope was weakly associated with ACPA-negative RA, but no dosage effect was detected (p=0.016, OR=1.17, 95% CI 1.03 to 1.34). A combination of HLA-DRB1*12:01 and DRB1*09:01 showed a strong association with susceptibility to ACPA-negative RA (p=0.00013, OR=3.62, 95% CI 1.79 to 7.30). Homozygosity for HLA-DR8 was significantly associated with ACPA-negative RA (p=0.0070, OR=2.16, 95% CI 1.22 to 3.82). It was also found that HLA-DRB1*15:02 and *13:02 were protective against ACPA-negative RA (p=0.00010, OR=0.68, 95% CI 0.56 to 0.83 and p=0.00059, OR=0.66, 95% CI 0.52 to 0.84, respectively). Conclusions In this large-scale association study multiple alleles and diplotypes were found to be associated with susceptibility to, or protection against, ACPA-negative RA.

ACPA-Negative RA Consists of Two Genetically Distinct Subsets Based on RF Positivity in Japanese

HLA-DRB1, especially the shared epitope (SE), is strongly associated with rheumatoid arthritis (RA). However, recent studies have shown that SE is at most weakly associated with RA without anti-citrullinated peptide/protein antibody (ACPA). We have recently reported that ACPA-negative RA is associated with specific HLA-DRB1 alleles and diplotypes. Here, we attempted to detect genetically different subsets of ACPA-negative RA by classifying ACPA-negative RA patients into two groups based on their positivity for rheumatoid factor (RF). HLA-DRB1 genotyping data for totally 954 ACPA-negative RA patients and 2,008 healthy individuals in two independent sets were used. HLA-DRB1 allele and diplotype frequencies were compared among the ACPA-negative RF-positive RA patients, ACPA-negative RF-negative RA patients, and controls in each set. Combined results were also analyzed. A similar analysis was performed in 685 ACPA-positive RA patients classified according to their RF positivity. As a result, HLA-DRB1*04:05 and *09:01 showed strong associations with ACPA-negative RF-positive RA in the combined analysis (p = 8.8×10−6 and 0.0011, OR: 1.57 (1.28–1.91) and 1.37 (1.13–1.65), respectively). We also found that HLA-DR14 and the HLA-DR8 homozygote were associated with ACPA-negative RF-negative RA (p = 0.00022 and 0.00013, OR: 1.52 (1.21–1.89) and 3.08 (1.68–5.64), respectively). These association tendencies were found in each set. On the contrary, we could not detect any significant differences between ACPA-positive RA subsets. As a conclusion, ACPA-negative RA includes two genetically distinct subsets according to RF positivity in Japan, which display different associations with HLA-DRB1. ACPA-negative RF-positive RA is strongly associated with HLA-DRB1*04:05 and *09:01. ACPA-negative RF-negative RA is associated with DR14 and the HLA-DR8 homozygote.

Myelin basic protein as a novel genetic risk factor in rheumatoid arthritis--a genome-wide study combined with immunological analyses.

Rheumatoid arthritis (RA) is a major cause of adult chronic inflammatory arthritis and a typical complex trait. Although several genetic determinants have been identified, they account for only a part of the genetic susceptibility. We conducted a genome-wide association study of RA in Japanese using 225,079 SNPs genotyped in 990 cases and 1,236 controls from two independent collections (658 cases and 934 controls in collection1; 332 cases and 302 controls in collection2), followed by replication studies in two additional collections (874 cases and 855 controls in collection3; 1,264 cases and 948 controls in collection4). SNPs showing p<0.005 in the first two collections and p<10−4 by meta-analysis were further genotyped in the latter two collections. A novel risk variant, rs2000811, in intron2 of the myelin basic protein (MBP) at chromosome 18q23 showed strong association with RA (p = 2.7×10−8, OR 1.23, 95% CI: 1.14–1.32). The transcription of MBP was significantly elevated with the risk allele compared to the alternative allele (p<0.001). We also established by immunohistochemistry that MBP was expressed in the synovial lining layer of RA patients, the main target of inflammation in the disease. Circulating autoantibody against MBP derived from human brain was quantified by ELISA between patients with RA, other connective tissue diseases and healthy controls. As a result, the titer of anti-MBP antibody was markedly higher in plasma of RA patients compared to healthy controls (p<0.001) and patients with other connective tissue disorders (p<0.001). ELISA experiment using citrullinated recombinant MBP revealed that a large fraction of anti-MBP antibody in RA patients recognized citrullinated MBP. This is the first report of a genetic study in RA implicating MBP as a potential autoantigen and its involvement in pathogenesis of the disease.

Incidence of hepatitis B virus reactivation in patients with resolved infection on immunosuppressive therapy for rheumatic disease: a multicentre, prospective, observational study in Japan

Background Although the reactivation of hepatitis B virus (HBV) is recognised as a serious complication in patients with rheumatic disease (RD) receiving immunosuppressive drugs (ISDs), the incidence and risk factors for reactivation remain controversial. Objectives To investigate the incidence and risk factors for HBV reactivation in patients with RD. Methods We performed a multicentre, observational, prospective study over 2 years in patients with resolved HBV infection. Patients with RD treated with a dose of ≥5 mg/day prednisolone and/or synthetic or biological ISDs with negative HB virus surface antigen and positive anti-HB virus surface antibody (HBsAb) and/or anti-HB virus core antibody (HBcAb) were enrolled. Quantitative HBV DNA results and related data were regularly recorded. Results Among 1042 patients, including 959 with rheumatoid arthritis, HBV DNA was detected in 35 (1.93/100 person-years), with >2.1 log copies/mL observed in 10 patients (0.55/100 person-years). None of the reactivated patients, including seven treated with a nucleic acid analogue, showed overt hepatitis. Low HBsAb titres and advanced age seemed to be risk factors for HBV reactivation; however, reactivation was observed in three patients with positive HBsAb and negative HBcAb test results. The risk of reactivation was lower with methotrexate but higher with prednisolone among the different types of ISDs. The intervals from the start of ISD to reactivation were relatively long (3–182 months; median, 66 months). Conclusions The incidence of HBV reactivation with ISD use was 1.93/100 person-years in patients with RD with resolved HBV infection. No overt hepatitis was observed in the reactivated patients.

An Association Between Amino Acid Position 74 of HLA–DRB1 and Anti–Citrullinated Protein Antibody Levels in Japanese Patients With Anti–Citrullinated Protein Antibody–Positive Rheumatoid Arthritis

Anti–citrullinated protein antibodies (ACPAs) are highly specific to rheumatoid arthritis (RA), and strong associations between HLA–DRB1 alleles and ACPA levels have been detected in RA patients. We undertook this study to elucidate the associations between particular amino acid positions in HLA–DRB1 and ACPA levels in patients with RA.

Drug retention and discontinuation reasons between seven biologics in patients with rheumatoid arthritis -The ANSWER cohort study-

The purpose of this study was to evaluate the retention and discontinuation reasons of seven biological disease-modifying antirheumatic drugs (bDMARDs) in a real-world setting of patients with rheumatoid arthritis (RA). 1,037 treatment courses with bDMARDs from 2009 to 2016 [female, 81.8%; baseline age, 59.6 y; disease duration 7.8 y; rheumatoid factor positivity 81.5%; Disease Activity Score in 28 joints using erythrocyte sedimentation rate (DAS28-ESR), 4.4; concomitant prednisolone 43.5% and methotrexate 68.6%; Bio-naïve, 57.1%; abatacept (ABT), 21.3%; tocilizumab (TCZ), 20.7%; golimumab (GLM), 16.9%; etanercept (ETN), 13.6%; adalimumab (ADA), 11.1%; infliximab (IFX), 8.5%; certolizumab pegol (CZP), 7.9%] were included in this multi-center, retrospective study. Drug retention and discontinuation reasons at 36 months were estimated using the Kaplan-Meier method and adjusted by potent confounders using Cox proportional hazards modeling. As a result, 455 treatment courses (43.9%) were stopped, with 217 (20.9%) stopping due to inefficacy, 113 (10.9%) due to non-toxic reasons, 86 (8.3%) due to toxic adverse events, and 39 (3.8%) due to remission. Drug retention rates in the adjusted model were as follows: total retention (ABT, 60.7%; ADA, 32.7%; CZP, 43.3%; ETN, 51.9%; GLM, 45.4%; IFX, 31.1%; and TCZ, 59.2%; P < 0.001); inefficacy (ABT, 81.4%; ADA, 65.7%; CZP, 60.7%; ETN, 71.3%; GLM, 68.5%; IFX, 65.0%; and TCZ, 81.4%; P = 0.015), toxic adverse events (ABT, 89.8%; ADA, 80.5%; CZP, 83.9%; ETN, 89.2%; GLM, 85.5%; IFX, 75.6%; and TCZ, 77.2%; P = 0.50), and remission (ABT, 95.5%; ADA, 88.1%; CZP, 91.1%; ETN, 97.5%; GLM, 94.7%; IFX, 86.4%; and TCZ, 98.4%; P < 0.001). In the treatment of RA, ABT and TCZ showed higher overall retention, and TCZ showed lower inefficacy compared to IFX, while IFX showed higher discontinuation due to remission compared to ABT, ETN, GLM, and TCZ in adjusted modeling.

SAT0140 Prevalence of Reactivation of Hepatitis B Virus (HBV) in Patients with Resolved Hbv Hepatitis on Immunosuppressive Therapy for Rheumatic Disease: Multicentre Prospective Observational Study in Japan

Background Although the reactivation of hepatitis B virus (HBV) is recognized as a serious complication in patients with connective tissue disease (CTD) receiving immunosuppressive drugs (ISDs), the incidence and risk factors of reactivation remain controversial. Objectives We performed a multicentre, observational, prospective study across 16 Japanese Red Cross hospitals to clarify the prevalence and pathophysiology of HBV reactivation in patients with CTD. We report the results of 2-year observations in patients with resolved HBV infection. Methods Patients with CTD, treated with a dose of ≥5 mg/day prednisolone, and/or synthetic or biological ISDs, with negative HBs antigen and positive anti HBs and/or HBc antibody (Ab), were enrolled. HBV-DNA (RT-PCR) and related data were registered regularly. Results Among 1040 patients, including 950 rheumatoid arthritis, HBV-DNA was detected in 32 patients (1.75/100 person-years), and >2.1 log copy/ml was observed in 9 cases (0.49/100 person-years) over 1–2 years of observation. None of the reactivated patients, including six treated with a nucleic acid analogue, showed overt hepatitis. Low HBs Ab titres and advanced age seemed to be risk factors for HBV reactivation; however, reactivation was observed in 3 patients with positive HBs Ab and negative HBc Ab. The risk of reactivation among the different types of ISDs was not statistically different. The intervals from the start of ISD to reactivation were longer than previous reports (4–157 months, average 58.9 months). Conclusions HBV reactivation with ISD was 1.75/100 person-years in CTD patients with resolved HBV infection. Overt hepatitis was observed in none of the reactivated patients, and low HBs antibody titres and advanced age were risk factors for reactivation. Patients with positive HBs Ab and negative HBc Ab are not free from the risk of reactivation of HBV. References B. J Hepatol 2012: 57. 167–185 Disclosure of Interest None declared

Suppressor of TCR signaling-2 (STS-2) suppresses arthritis development in mice

Abstract Objectives: Suppressor of TCR signaling-2 (STS-2) is one of the RA susceptibility genes identified in genome-wide association studies (GWAS). We tried to verify the involvement of STS-2 on the development of autoimmune arthritis in a mouse model. Methods: STS-2 knock-out (KO) and wild type (WT) mice were immunized with chicken type II collagen (CII). For CD4+ helper T cell (Th) subset analysis, intracellular cytokines in splenocytes and lymph node cells were stained and analyzed by flow cytometry. Regulatory T cell (Treg) function was analyzed by co-culturing effector CD4+T cells and Tregs collected from non-immunized mice. Results: CII-immunized STS-2 KO mice developed arthritis more frequently than WT mice. Although the T cell activation profile and Th subset in spleen and LNs were similar between STS-2 KO and WT mice, STS-2 KO mice showed increased IL-2-producing CD4+T cells in spleen when compared with WT mice. Accordingly, STS-2 KO CD4+T cells promoted IL-2 production by TCR stimulation. However, STS-2 KO Tregs normally suppressed T cell proliferation. Conclusion: We proved that STS-2 is involved in the arthritis development by collagen-induced arthritis. Higher IL-2 production from STS-2 KO T cells is suggested to have a main pathogenic role in arthritis development.