RF-negative Rheumatoid Arthritis Research Articles

Therapeutic Potential of a Novel Bifidobacterium Identified Through Microbiome Profiling of RA Patients With Different RF Levels.

The potential therapeutic effects of probiotic bacteria in rheumatoid arthritis (RA) remain controversial. Thus, this study aimed to discover potential therapeutic bacteria based on the relationship between the gut microbiome and rheumatoid factor (RF) in RA. Bacterial genomic DNA was extracted from the fecal samples of 93 RA patients and 16 healthy subjects. Microbiota profiling was conducted through 16S rRNA sequencing and bioinformatics analyses. The effects of Bifidobacterium strains on human peripheral blood mononuclear cells and collagen-induced arthritis (CIA) mice were assessed. Significant differences in gut microbiota composition were observed in patients with different RF levels. The relative abundance of Bifidobacterium and Collinsella was lower in RF-high than in RF-low and RF-negative RA patients, while the relative abundance of Clostridium of Ruminococcaceae family was higher in RF-high than in RF-low and RF-negative patients. Among 10 differentially abundant Bifidobacterium, B. longum RAPO exhibited the strongest ability to inhibit IL-17 secretion. Oral administration of B. longum RAPO in CIA mice, obese CIA, and humanized avatar model significantly reduced RA incidence, arthritis score, inflammation, bone damage, cartilage damage, Th17 cells, and inflammatory cytokine secretion. Additionally, B. longum RAPO significantly inhibited Th17 cells and Th17-related genes—IL-17A, IRF4, RORC, IL-21, and IL-23R—in the PBMCs of rheumatoid arthritis patients. Our findings suggest that B. longum RAPO may alleviate RA by inhibiting the production of IL-17 and other proinflammatory mediators. The safety and efficacy of B. longum RAPO in patients with RA and other autoimmune disorders merit further investigation.

THU0181 SOLUBLE VASCULAR BIOMARKERS IN RHEUMATOID ARTHRITIS AND ANKYLOSING SPONDYLITIS: EFFECTS OF ONE-YEAR ANTI-TNF-Α THERAPY

Background:Rheumatoid arthritis (RA) and ankylosing spondylitis (AS) have been associated with inflammatory atherosclerosis, increased cardiovascular (CV) morbidity and mortality. Numerous proteins may serve as biomarkers of inflammatory atherosclerosis. The treatment of arthritis by tumour necrosis factor α (TNF-α) inhibitors may decrease the serum concentrations of these biomarkers.Objectives:In this study we wished to determine circulating levels of oxidized LDL (oxLDL) - β2 glycoprotein I (β2GPI) complexes (AtherOx), anti-hsp60 antibodies, soluble urokinase plasminogen activator receptor (sUPAR) and N-terminal B-type natriuretic peptide (NT-proBNP) in sera of RA and AS patients. We also wished to assess the effects of anti-TNF treatment on these biomarkers.Methods:Altogether 53 arthritis patients including 36 RA patients treated with either etanercept (ETN) or certolizumab pegol (CZP) and 17 AS patients treated with ETN were included in a 12-month follow-up study.Circulating oxLDL/β2gpI complexes, anti-human Hsp60 immunoglobulin G (IgG) levels and BNP8-29fragment levels were assessed by ELISA. suPAR levels were assessed by suPARnostic®Quick Triage test. All laboratory assessments were performed at baseline, as well as 6 and 12 months after treatment initiation. Results were associated with DAS28, BASDAI, CRP.Results:In the mixed cohort of 53 arthritis patients, the circulating levels of oxLDL/β2gpI significantly decreased after 12 months of anti-TNF therapy (0.20±0.11 U/ml) compared to baseline (0.24±0.10 U/ml; p=0.014). There was a tendency of non-significant decrease after 6 months (0.23±0.14 U/ml) versus baseline. Anti-Hsp60 antibody levels did not change after 6 months (158.6±138.6 AU/ml) and 12 months (167.3±143.3 AU/ml) compared to baseline (170.3±140.4 AU/ml). Among the patients, 21.2% had low, 36.4% “observe”, 9.1% high and 33.3% critical suPAR levels. suPAR levels showed a tendency of non-significant decrease after 6 months (11.3±17.7 ng/ml) and 12 months (10.3±15.3 ng/ml) versus baseline (11.5±16.4 ng/ml). However, when the four serum level categories described above were considered, suPAR concentrations exerted significant decrease in RA patients with critical suPAR levels (>9ng/ml) (p=0.04). Similarly, BNP fragment levels showed only a tendency of decrease after 6 months (518.2±422.4 pmol/l) and 12 months (484.1±418.2 pmol/l) versus baseline (530.8±441.8 pmol/l). However, serum BNP levels at baseline and after 12 months were significantly increased in CCP positive compared to CCP negative RA patients (baseline: 670.6±323.0 versus 138.0±436.4 pmol/l; p=0.030 and 12 months: 652.9±283.2 versus 456.5±423.1 pmol/l; p=0.021), as well as in RF positive compared to RF negative RA patients (baseline: 680.6±381.6 versus 292.9±198.3 pmol/l; p=0.007 and 12 months: 668.9±346.5 versus 312.2±207.0 pmol/l; p=0.001).Conclusion:One-year anti-TNF therapy significantly decreased circulating oxLDL/β2gpI complex levels. This therapy also decreased suPAR levels in patients with critically high suPAR. BNP fragment levels were associated with seropositivity in RA. These vascular biomarkers may reflect the effects of TNF inhibition on endothelial activation.Acknowledgments:This study was sponsored by an investigator-initiated grant from Pfizer.Disclosure of Interests:Anita Pusztai: None declared, Attila Hamar: None declared, Ágnes Horváth: None declared, Edit Végh: None declared, Nóra Bodnár: None declared, György Kerekes: None declared, Monika Czókolyová: None declared, Szilvia Szamosi: None declared, Levente Bodoki: None declared, Katalin Hodosi: None declared, Andrea Domjan: None declared, Gábor Nagy: None declared, Ibolya Szöllösi: None declared, Luis Lopez Employee of: Retired employee of Corgenix Inc., Eiji Matsuura: None declared, Zoltán Prohászka: None declared, Sándor Szántó: None declared, Zoltán Nagy: None declared, Yehuda Shoenfeld: None declared, Zoltán Szekanecz Grant/research support from: Pfizer, UCB, Consultant of: Sanofi, MSD, Abbvie, Pfizer, Roche, Novertis, Lilly, Gedeon Richter, Amgen, Gabriella Szücs: None declared

Is the epidemiology of rheumatoid arthritis changing? Results from a population-based incidence study, 1985–2014

ObjectivesTo examine trends in the incidence of rheumatoid arthritis (RA) from 2005 to 2014 overall and by serological status as compared with 1995–2004 and 1985–1994.MethodsWe evaluated RA incidence trends in...

Mediterranean diet and risk of rheumatoid arthritis: a population-based case-control study

BackgroundThe Mediterranean diet has been associated with lower mortality and lower risk of cardiovascular diseases and cancer. Although its components have been analysed in several studies, only one study has specifically investigated the association between Mediterranean diet and risk of rheumatoid arthritis (RA), and reported no association.MethodsData on 1721 patients with incident RA (cases) and 3667 controls, matched on age, gender and residential area, from the Swedish epidemiological investigation of RA (EIRA), a population-based case-control study, were analysed using conditional logistic regression. The Mediterranean diet score, ranging from 0 to 9, was calculated from a 124-item food frequency questionnaire.ResultsIn the EIRA study (median age of participants 53 years), 24.1% of the patients and 28.2% of the controls had high adherence to the Mediterranean diet (a score between 6 and 9). After adjustments for body mass index, educational level, physical activity, use of dietary supplements, energy intake, and smoking, high adherence reduced the odds of developing RA by 21% (OR 0.79; 95% CI 0.65–0.96) as compared to low adherence (a score between 0 and 2). The OR was even lower among men (OR 0.49; 95% CI 0.33–0.73), but no significant association was found among women (OR 0.94; 95% CI 0.74–1.18). An association between high diet score and low risk of RA was observed in rheumatoid factor (RF)-positive (OR 0.69; 95% CI 0.54–0.88), but not RF-negative RA (OR 0.96; 95% CI 0.68–1.34), and in RA characterised by presence of antibodies to citrullinated peptides (ACPA), but not in ACPA-negative RA.ConclusionsIn this large population-based case-control study, the Mediterranean diet score was inversely associated with risk of RA. However, an association was only found among men and only in seropositive RA.

Interleukin-34 in the Serum and Synovial Fluid of Rheumatoid Arthritis Patients: Relation to Disease Activity and Radiographic Damage

Abstract Background Interleukin 34 (IL-34) is a recently discovered proinflammatory cytokine that can promote inflammation and osteoclastogenesis in arthritic joints. In this study, we tried to assess the serum and synovial fluid (SF) levels of IL-34 in rheumatoid arthritis (RA) patients, and to determine its relationship with disease activity and radiographic damage. Patients and methods ELISA was used to evaluate IL-34 levels in the serum of RA patients (n=50), osteoarthritis (OA) patients (n=28), and healthy control subjects (n=20) and in SF isolated from RA and OA patients. Disease activity in RA patients was assessed using the disease activity score-28 (DAS 28). The extent of radiographic joint damage, narrowing, and erosions was assessed. Results Serum IL-34 level was significantly elevated in RA patients compared to that in OA patients and healthy controls (P<0.0001). Synovial IL-34 level was also significantly elevated in RA patients compared to that in OA patients (P=0.0004). Serum and synovial IL-34 levels were significantly higher in cases that were rheumatoid factor (RF) positive (n=28) compared to the levels in RF-negative RA cases (n=22) (P=0.03 and P=0.04, respectively). Serum and synovial IL-34 levels were positively correlated with RF (r=0.43, P=0.02 and r=0.39, P=0.03, respectively), and with the extent of radiological damage (SENS) (P=0.0002 and P<0.0001, respectively). However, no significant correlation between IL-34 levels and disease activity was found. Conclusion IL-34 appears to play a key role in the pathogenesis of RA and contribute to bone destruction, making it a potential therapeutic target in the management armamentarium of the disease.

Influence of Cigarette Smoking on Rheumatoid Arthritis Risk in the Han Chinese Population.

Cigarette smoking has been shown in European populations to be associated with rheumatoid arthritis (RA) susceptibility. This study aims to examine the association of smoking with RA in the Han Chinese population. 718 Han Chinese RA patients and 404 healthy controls were studied. The associations of cigarette smoking (current, former or ever vs. never smokers, and pack-years of exposure) with RA, anti-cyclic citrullinated peptide antibody (ACPA) positive RA, IgM rheumatoid factor (RF) positive RA, and baseline radiographic erosions (modified van der Heijde-Sharp scores) were assessed. The interaction between smoking and the HLA-DRB1 shared epitope (SE) in RA was also examined. In this study, 11 (1.53%) cases and 6 (1.49%) controls were former smokers (p = 0.95), while 95 (13.23%) cases and 48 (11.88%) controls were current smokers (p = 0.52). Trends toward associations between smoking status (ever vs. never) with RA-overall (p = 0.15, OR = 1.44), ACPA-positive RA (p = 0.24, OR = 1.37), RF-positive RA (p = 0.14, OR = 1.46), or the presence of radiographic erosions (p = 0.66, OR = 1.28) were observed although individually here were not statistically significant. There was no evidence of statistical interaction between smoking status (ever vs. never) and SE for all RA, ACPA-positive RA, ACPA-negative RA, RF-positive RA, RF-negative RA (p = 0.37, 0.50, 0.24, 0.26, and 0.81 respectively), and the 95% CI for the attributable proportion for all interactions included 0. This is the first study to examine the association of cigarette smoking with RA in the Han Chinese population. This study shows a trend toward an interaction between smoking and SE carriage influencing the risk of RA, though findings were not statistically significant. It is possible that in the presence of universal exposure to heavy air pollution the effect of smoking on RA risk may be obscured.

Calprotectin levels in rheumatoid arthritis and their correlation with disease activity: a meta-analysis

ABSTRACTObjective: We evaluated the relationship between calprotectin levels and rheumatoid arthritis (RA), and the correlation between plasma/serum calprotectin and RA activity.Methods: We searched PUBMED, EMBASE, and Cochrane databases and performed meta-analyses comparing plasma/serum or synovial fluid calprotectin levels in RA patients and controls, and correlation coefficients between calprotectin levels and disease activity for 28 joints (DAS28) as well as C-reactive protein (CRP) in RA patients.Results: Sixteen studies including 849 RA patients and 266 controls were available for meta-analysis. Meta-analysis showed that calprotectin levels were significantly higher in the RA group than in the control group (SMD = 2.337, 95% CI = 1.544–3.130, p < 1.0 × 10–8). Stratification by rheumatoid factor (RF) status revealed significantly elevated calprotectin levels in the RF-positive RA group compared to that of the RF-negative RA group (SMD = 0.574, 95% CI = 0.345–0.804, p = 9.2 × 10–7). Meta-analysis of correlation coefficients identified a significant positive correlation between calprotectin levels and CRP or DAS28 (correlation coefficient for CRP = 0.566, 95% CI = 0.512–0.615, p < 1.0 × 10–8; correlation coefficient for DAS28 = 0.438, 95% CI = 0.269–0.518, p = 2.5 × 10–6). Calprotectin levels in synovial fluid were significantly higher in the RA group than in the control group (SMD = 2.891, 95% CI = 1.067–4.715, p = 0.002).Conclusions: Our meta-analysis demonstrates that circulating and synovial fluid calprotectin levels are high in patients with RA, and that circulating calprotectin levels positively correlate with RA activity.

Only rheumatoid factor-positive subset of anti-citrullinated peptide/protein antibody-negative rheumatoid arthritis may seroconvert to anti-citrullinated peptide/protein antibody-positive.

Anti-citrullinated peptide/protein antibody (ACPA) has been reported to occur in about 60% of patients with early rheumatoid arthritis (RA), and about 80% in patients with established RA. While ACPA seroconversion is possible, previous reports have shown that it rarely occurs. We retrospectively determined the proportion of patients who underwent ACPA seroconversion and described the clinical characteristics of these cases. ACPA-negative RA patients who had undergone ACPA assessment more than once with an interval of 3 months or longer were investigated for ACPA seroconversion. The clinical characteristics of seroconverted patients were assessed. In 149 ACPA-negative RA patients, only eight patients (5.4%) converted to ACPA-positive during follow-up. We found that all eight of the seroconverted cases were positive for rheumatoid factor (RF) and showed bone erosions by X-ray. Of 56 ACPA-negative RF-positive RA patients, 14.3% of them seroconverted to ACPA-positive. None of the ACPA-negative RF-negative RA patients seroconverted to ACPA-positive. The proportion of total RA patients who experienced seroconversion from ACPA-negative to ACPA-positive was 5.4%. When ACPA-negative RA patients were subdivided into RF-negative and RF-positive subsets, only the RF-positive subset seroconverted to ACPA-positive. These results imply that RF-negative and RF-positive patients are distinct subsets within ACPA-negative RA patients.

Identification of IRX1 as a Risk Locus for Rheumatoid Factor Positivity in Rheumatoid Arthritis in a Genome-Wide Association Study.

Rheumatoid factor (RF) is a well-established diagnostic and prognostic biomarker in rheumatoid arthritis (RA). However, ∼20% of RA patients are negative for this anti-IgG antibody. To date, only variation at the HLA-DRB1 gene has been associated with the presence of RF. This study was undertaken to identify additional genetic variants associated with RF positivity. A genome-wide association study (GWAS) for RF positivity was performed using an Illumina Quad610 genotyping platform. A total of 937 RF-positive and 323 RF-negative RA patients were genotyped for >550,000 single-nucleotide polymorphisms (SNPs). Association testing was performed using an allelic chi-square test implemented in Plink software. An independent cohort of 472 RF-positive and 190 RF-negative RA patients was used to validate the most significant findings. In the discovery stage, a SNP in the IRX1 locus on chromosome 5p15.3 (SNP rs1502644) showed a genome-wide significant association with RF positivity (P = 4.13 × 10(-8) , odds ratio [OR] 0.37 [95% confidence interval (95% CI) 0.26-0.53]). In the validation stage, the association of IRX1 with RF was replicated in an independent group of RA patients (P = 0.034, OR 0.58 [95% CI 0.35-0.97] and combined P = 1.14 × 10(-8) , OR 0.43 [95% CI 0.32-0.58]). To our knowledge, this is the first GWAS of RF positivity in RA. Variation at the IRX1 locus on chromosome 5p15.3 is associated with the presence of RF. Our findings indicate that IRX1 and HLA-DRB1 are the strongest genetic factors for RF production in RA.

Human leukocyte antigen-G polymorphism influences the age of onset and autoantibody status in rheumatoid arthritis.

The study was conducted to investigate the frequency of three gene polymorphisms in the 3'-untranslated region (3'-UTR) of human leucocyte antigen-G (HLA-G) gene in south Indian patients with rheumatoid arthritis (RA) and analyze their influence on disease susceptibility, phenotype and treatment response. HLA-G 14 bp insertion (Ins)/deletion (del) (rs66554220), HLA-G +3142G>C (rs1063320) and +3187A>G (rs9380142) polymorphism was analyzed in 221 RA patients and 200 healthy controls. Frequency of HLA-G genotypes or alleles did not differ between patients and controls. Analysis based on rheumatoid factor (RF) status revealed that the frequency of allele 'A' (rs9380142) was significantly higher in RF-positive than in RF-negative patients [84% vs 74%, Yates-corrected P value (Pc) = 0.04, odds ratio (OR) = 1.8, 95% confidence interval (CI) = 1.0-3.2]. A similar difference was maintained in RF-positive female patients than their RF-negative counterparts (83% vs 71%, Pc = 0.02, OR = 1.9, 95% CI = 1.0 to 3.4) and between RF-positive and RF-negative young onset RA (YORA) patients (84% vs 73%, Pc = 0.03, OR = 1.9, 95% CI = 1.0-3.2), suggesting that rs9380142 polymorphism influenced RF status. The 14 bp Ins allele of rs66554220 was significantly more prevalent in RF-positive YORA than in RF-positive late onset RA (LORA) patients (51% vs 25%, P = 0.03, OR = 3.1, 95% CI = 1.1-9.8). Frequency of the four major haplotypes [InsGA (48%), DelGA (22%), DelCG (18%), DelCA (9.7%)] observed did not differ between cases and controls. HLA-G does not appear to be a risk factor for development of RA in south Indian Tamils but may act as a genetic modifier of clinical phenotype in terms of autoantibody production, gender preference and age at disease onset.

Familial aggregation of arthritis-related diseases in seropositive and seronegative rheumatoid arthritis: a register-based case-control study in Sweden

ObjectivesOur objective was to estimate the risk of developing rheumatoid arthritis (RA) associated with a family history of non-RA arthritis-related diseases. This familial co-aggregation is of clinical interest since it...

THU0462 Anti-Citrullinated Peptide/Protein Antibody (ACPA)-Negative RA Shares Large Proportion of Susceptibility Genes with Acpa-Positive RA

BackgroundSusceptibility genes for anti-citrullinated peptide/protein antibodies (ACPA)-positive rheumatoid arthritis (RA) have been successfully discovered by the development of genome-wide association studies (GWAS). However, little is known about the genetic background...

A1.29 In rheumatoid arthritis, smoking is not primarily associated with anti-citrullinaged protein antibodies, but with the presence of several autoantibodies

Background and ObjectivesIn rheumatoid arthritis (RA), smoking has been described to be specifically associated with the presence of anti-citrullinated protein antibodies (ACPA). However, smoking has also been shown to be...

Association between functional Fc receptor-like 3 (FCRL3) −169 C/T polymorphism and susceptibility to seropositive rheumatoid arthritis in Asians: A meta-analysis

The aim of this study was to determine whether the functional Fc receptor like-3 (FCRL3) -169 C/T polymorphism confers susceptibility to rheumatoid arthritis (RA). A meta-analysis was conducted on the associations between the FCRL3 -169 C/T polymorphism and RA. A total of 17 comparison studies including 11,170 patients and 11,142 controls were considered in the meta-analysis. The meta-analysis showed no association between RA and the FCRL3 -169 C allele in study subjects (OR = 1.046, 95% CI = 0.997-1.098, p = 0.068). Stratification by ethnicity indicated an association between the FCRL3 -169 C allele and RA in Asians (OR = 1.101, 95% CI = 1.035-1.174, p = 0.002), but not in Europeans. Stratification of patients according to the presence of rheumatoid factor (RF) revealed a different significant association between the C allele and RA in RF-positive and RF-negative RA patients. Stratification by ethnicity indicated an association between the FCRL3 -169 C allele and RF-positive RA in Asians (OR = 1.093, 95% CI = 1.004-1.189, p = 0.040), but not in Europeans. This meta-analysis demonstrates that the FCRL3 -169 C/T polymorphism may confer susceptibility to seropositive RA in Asians.

OP0190 Smoking is Not Primarily Associated with ACPA, but with the Presence of Multiple Autoantibodies in RA

BackgroundSmoking is associated with the presence of autoantibodies in various diseases, such as chronic obstructive pulmonary disease (COPD)1, systemic lupus erythematosus (SLE)2 and polymyositis3. In rheumatoid arthritis (RA), smoking is...

AB0113 Association between functional fc receptor-like 3 (fcrl3) -169 c/t polymorphism and susceptibility to seropositive rheumatoid arthritis: a meta-analysis

BackgroundThe Fc receptor-like 3 gene (FCRL3) is located on chromosome 1q21, a region implicated in susceptibility to several autoimmune diseases by whole-genome linkage studies. The FCRL3 protein is an orphan...

SAT0002 ACPA-negative rheumatoid arthritis consists of two genetically distinct subsets based on RF positivity

BackgroundHLA-DRB1, especially the shared epitope (SE), is strongly associated with rheumatoid arthritis (RA) [1]. However, recent studies have shown that SE is at most weakly associated with RA without anti-citrullinated...

Superior performance of the CCP3.1 test compared to CCP2 and MCV in the rheumatoid factor-negative RA population

Anti-citrullinated protein/peptide antibodies (ACPAs) have recently been identified as sensitive and specific diagnostic and prognostic markers in rheumatoid arthritis (RA). In this study, we wished to assess the diagnostic performance of the third-generation anti-CCP3.1 assay, but with special focus on the rheumatoid factor (RF)-negative RA population. Anti-CCP as well as anti-MCV was tested in 119 RA patients and 118 control patients using second and third-generation assays. Using these optimal cut-off levels, the diagnostic sensitivity of anti-CCP2, CCP3, and CCP3.1 was 74.8, 78.8, and 83.0%, respectively, while the specificity was 95.7, 96.6, and 98.3%, respectively. The diagnostic performance of the CCP3.1 test was significantly better than that of CCP2 (p = 0.041). In addition, the CCP3.1 test performed significantly better than the MCV test as well (p = 0.0003). When the diagnostic performance of the CCP3.1, CCP2, and MCV tests was compared in the 35 RF-negative patients, the CCP3.1 test exerted significantly better performance than the MCV test (p = 0.006), and it also showed a tendency of better performance in comparison with the CCP2 test (p = 0.131). In conclusion, the CCP3.1 assay can significantly increase the sensitivity of ACPA testing in RF-negative RA, as well as in the total RA population.

Performance of hands and feet radiographs in differentiation of psoriatic arthritis from rheumatoid arthritis

The purpose of this study was to determine useful radiographic findings for differentiating psoriatic arthritis (PsA) from rheumatoid factor (RF)-positive or -negative rheumatoid arthritis (RA) in Japanese patients. We accrued 85 patients with PsA. Controls included 135 patients with RA (85 RF-positive, 50 RF-negative) matched for gender and disease duration with PsA patients. Radiographs of hands and feet were obtained, and distal interphalangeal (DIP) erosive disease, joint osteolysis, tuft osteolysis, juxta-articular bony proliferation (JBP), periosteal new bone formation and bony ankylosis, which were identified using the definitions developed by an earlier study, were compared between the PsA and RA groups. For radiographic features of hands, the frequencies of JBP, periosteal new bone, and diffuse soft tissue swelling of the fingers were significantly higher in PsA patients than in RF-positive RA patients. However, only the frequency of JBP significantly differed between PsA and RF-negative RA patients. In feet, the frequencies of DIP erosive disease, tuft osteolysis, JBP, and diffuse soft tissue swelling of the toes were significantly higher in PsA patients than in RF-positive RA patients. However, only the frequency of JBP significantly differed between PsA and RF-negative RA patients. JBP was the most important radiographic feature for discriminating PsA from both RF-positive and -negative RA, confirming the study by the CASPAR group that showed that JBP is the only radiologic feature that can discriminate PsA from other inflammatory arthritides. This study showed the utility of plain radiographs for diagnosis of PsA.

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.