Association of rheumatoid arthritis and its severity with human leukocytic antigen-DRB1 alleles in Kurdish region in North of Iraq

Background Rheumatoid arthritis is a complex multifactorial chronic disease, the importance of human leukocytic antigen (HLA) as a major genetic risk factor for rheumatoid arthritis was studied worldwide. The objective of this study is to identify the association of HLA-DRB1 subtypes with rheumatoid arthritis and its severity in Kurdish region. Methods A case–control study recruited 65 rheumatoid arthritis patients and 100 healthy individuals as control group all over the Kurdistan region/Iraq. Both patient and control groups are genotyped using polymerase chain reaction with sequence specific primer. Anti-CCP antibodies were measured by ELISA test. Rheumatoid factor, C-reactive protein, and disease activity score 28 which measured by DAS-28 values were calculated. The DAS-28 was used to assess the clinical severity of the patients. Results HLA-DRB1-0404 and HLA-DRB1-0405 frequencies showed a strong association with disease susceptibility (P < 0.001). The frequency of HLA-DRB1-0411 and HLA-DRB1-0413 were significantly higher in control group (P < 0.001). The frequency of rheumatoid factor and Anti-CCP were significantly higher among shared epitope-positive patients compared to shared epitope-negative patients (P < 0.001). Regarding the disease activity by DAS-28, rheumatoid arthritis patients didn’t show significant difference between the shared epitope-positive and shared epitope-negative patients. Conclusions HLA-DR0404 and HLA-DR0405 alleles are related to RA, while HLA-DR1-0411 and HLA-DRB1-0413 protect against RA in the Kurdistan region in the North of Iraq.

Impact of the HLA-DRB1 shared epitope on responses to treatment with tofacitinib or abatacept in patients with rheumatoid arthritis

Objectives The aim of this study was to compare the clinical effectiveness of tofacitinib and abatacept and clarify the impact of the HLA-DRB1 shared epitope (SE) on responses to these treatments in patients with rheumatoid arthritis (RA). Methods After adjustments by propensity score matching, 70 out of 161 patients receiving tofacitinib and 70 out of 131 receiving abatacept were extracted. The clinical effectiveness of both drugs over 24 weeks and the impact of the copy numbers of SE on effectiveness outcomes were investigated. Results The percentage of patients in remission in the 28-joint count disease activity score using the erythrocyte sedimentation rate (DAS28-ESR) did not significantly differ between patients receiving tofacitinib and abatacept at week 24 (32% vs 37%, p = 0.359). The mean change at week 4 in DAS28-ESR from baseline was significantly greater in patients receiving tofacitinib than in those receiving abatacept (− 1.516 vs − 0.827, p = 0.0003). The percentage of patients in remission at week 4 was 30% with tofacitinib and 15% with abatacept (p = 0.016). When patients were stratified by the copy numbers of SE alleles, differences in these numbers did not affect DAS28-ESR scores of patients receiving tofacitinib. However, among patients receiving abatacept, DAS28-ESR scores were significantly lower in patients carrying 2 copies of SE alleles than in those carrying 0 copies at each time point throughout the 24-week period. Furthermore, the percentage of patients in remission with DAS28-ESR at week 24 was not affected by the copy numbers of SE alleles in patients receiving tofacitinib (p = 0.947), whereas it significantly increased as the copy numbers became higher in patients receiving abatacept (p = 0.00309). Multivariable logistic regression analyses showed a correlation between the presence of SE and DAS28-ESR remission in patients receiving abatacept (OR = 25.881, 95% CI = 3.140–213.351, p = 0.0025), but not in those receiving tofacitinib (OR = 1.473, 95% CI = 0.291–7.446, p = 0.639). Conclusions Although the clinical effectiveness of tofacitinib and abatacept was similar at week 24, tofacitinib was superior to abatacept for changes from baseline in DAS28-ESR and the achievement of remission at week 4. SE positivity was associated with the achievement of DAS28-ESR remission by week 24 in patients receiving abatacept, but not in those receiving tofacitinib.

OP0012 ASSOCIATION BETWEEN PASSIVE SMOKING IN CHILDHOOD AND ADULTHOOD, AND RHEUMATOID ARTHRITIS: RESULTS FROM THE FRENCH E3N-EPIC COHORT STUDY

Background:Rheumatoid arthritis (RA) is a systemic autoimmune disease of multifactorial aetiology, which preferentially affects women. To date, active smoking has been the most reproducibly reported risk factor for anti-citrullinated protein antibodies (ACPA) positive RA, particularly persons who carry the HLA-DRB1-shared epitope (SE) alleles.Objectives:We aimed to investigate the relationships between passive smoking in childhood (PSc) or in adulthood (PSa), and the risk of incident RA in a large prospective cohort of healthy French women.Methods:The E3N-EPIC (Etude Epidémiologique auprès des femmes de la Mutuelle générale de l’Education Nationale) is a French prospective cohort study that investigates environmental factors associated with chronic diseases. It follows 98,995 healthy French women since 1990 covered by a national health insurance primarily involving teachers. RA cases have been previously identified with specific questionnaires and medication reimbursement database. Women were considered exposed to PSc if they self-declared staying in a smoky room several hours a day during childhood, and to PSa if they self-declared being exposed at least one hour a day to passive smoking while adults. We used Cox multivariable regression models with age as the timescale (model 1), adjusted on smoking status (never, current, or former smoker) and on the two types of passive smoking (model 2), and on educational level, and BMI (model 3). Stratified analyses were conducted depending on the active smoking status (never or ever-smoker).Results:79,806 women were included in the study. Mean (± SD) age at cohort entry was 49.0 (± 6.4) years. Among them, 698 incident RA cases were identified, diagnosed after a mean of 11.7 (± 5.8) years after baseline. In the whole cohort, 10,810 (13.5%) women were exposed to PSc, 42,807 (53.6%) to PSa, 6,581 (8.25%) were exposed to both, and 47,036 (58.9%) were exposed to either.In the whole population, PSc was positively associated with the risk of RA in all three models (HR 1.24; 95% CI [1.01 to 1.51] in Model 3). In stratified analyses on smoking status, PSc was associated with RA among never-smoking women (HR 1.42; 95% CI [1.07 to 1.88]), but not among ever-smoking women (HR 1.10; 95% CI [0.83;1.46]).In the whole population, PSa was also positively associated with the risk of RA in all three models (HR 1.19; 95% CI [1.02 to 1.40] in Model 3). In stratified analyses on the smoking status, PSa was associated with an increased RA risk only among never-smoking women (HR 1.27; 95% CI [1.02 to 1.57]) and not among ever-smoking women (HR 1.16; 95% CI [0.93;1.44]).Conclusion:In this large population-based prospective cohort study of French women, we reported that passive exposure to smoking during childhood or adulthood increased the risk of RA. The association was principally observed among never smoking women. These results suggest that smoking by-products, whether actively or passively inhaled absorbed, could generate autoimmunity, at least towards antigens involved in RA pathogenesis.References:[1]Karlson EW, Chang S-C, Cui J, et al. Gene-environment interaction between HLA-DRB1 shared epitope and heavy cigarette smoking in predicting incident rheumatoid arthritis. Ann Rheum Dis 2010;69:54–60.[2]Seror R, Henry J, Gusto G, et al. PSc increases the risk of developing rheumatoid arthritis. Rheumatology 2019;58:1154–62.[3]Nguyen Y, Salliot C, Gusto G, et al. Improving accuracy of self-reported diagnoses of rheumatoid arthritis in the French prospective E3N-EPIC cohort: a validation study. BMJ Open 2019;9:e033536.Disclosure of Interests:None declared

POS0379 DETERMINING IN WHICH PRECLINICAL STAGE HLA-SHARED EPITOPE ALLELES AND SMOKING EXERT THEIR EFFECT IN THE DEVELOPMENT OF RHEUMATOID ARTHRITIS

Background:The HLA shared epitope (SE) and smoking are the best known genetic and environmental risk factors for rheumatoid arthritis (RA) development; however, at which pre-RA stage they exert their effect is unknown. The following stages are discerned: an asymptomatic stage in which autoimmune responses can develop, a symptomatic stage (clinically suspect arthralgia (CSA)), and development of clinically apparent inflammatory arthritis (IA). Studies in the general asymptomatic population revealed contrasting results on the associations between SE-alleles and smoking and the presence of anti-citrullinated protein antibodies (ACPA). Furthermore, studies on these risk factors in the symptomatic pre-RA phase are scarce and these data might teach us whether SE-alleles and smoking are involved in symptom development and/or progression to clinical arthritis.Objectives:We aimed to determine at which pre-RA stage SE and smoking exert their effect. In this respect, the analyses were focused on the presence of ACPA, but associations for other anti-modified protein antibodies (anti-carbamylated and anti-acetylated protein antibodies (anti-CarP and AAPA, respectively)) were also studied.Methods:Results from the literature on the association of SE and smoking with ACPA in the asymptomatic population were summarized in inverse-variance weighted meta-analyses. In addition 577 CSA-patients were studied. Associations of SE and smoking with IgG ACPA were studied at baseline (CSA-onset), to assess an effect on symptom development. Additionally, patients were monitored for the development of clinically apparent inflammatory arthritis (IA) for median 2 years and associations of SE, smoking and auto-antibodies with progression to IA were determined. Analyses were stratified for ACPA-status and associations in ACPA-positive patients were validated in meta-analyses with other arthralgia-cohorts. Finally analyses were repeated for anti-CarP and AAPA.Results:Meta-analyses showed that SE is not associated with ACPA-positivity in the asymptomatic population (OR 1.06 (95%CI 0.69-1.64)), whereas smoking was associated (OR 1.37 (1.15-1.63)). At CSA-onset, both SE and smoking associated with ACPA-positivity (OR 2.08 (1.24-3.49) and OR 2.41 (1.31-4.43), respectively). During follow-up of CSA-patients SE associated with IA-development (HR 1.86 (1.23-2.82)), in contrast to smoking. SE conferred risk for IA-development in ACPA-negative CSA-patients (HR 1.71 (0.99-2.96)) and in ACPA-positive patients (CSA-cohort HR 1.29 (0.67-2.47); meta-analysis three arthralgia-cohorts HR 1.52 (1.08-2.15)). Investigating the other autoantibodies revealed that SE and smoking were not associated with anti-CarP or AAPA-positivity at CSA-onset; longitudinally AAPA associated with progression to IA independent from ACPA and RF (HR 1.79 (1.02-3.16)), whilst anti-Carp did not.Conclusion:SE and smoking act in partly different pre-RA stages. Although SE does not associate with ACPA in the general population, it does mediate symptom-development and further progression to clinical arthritis. Smoking confers risk to development of ACPA and/or joint symptoms, but is not further involved in IA-development. The time-specific biologic pathways that are underlying need further exploration. These data enhance the understanding of the timing of key genetic and environmental risk factors in the trajectory of RA development.Disclosure of Interests:None declared

AB0055 AUTOIMMUNE RESPONSE AGAINST THE SHARED EPITOPE SEQUENCE IN RHEUMATOID ARTHRITIS

Background:Rheumatoid Arthritis (RA) is a systemic autoimmune disease, associated with hiperproduction of autoantibodies (AAb), in which the most specific are the AAb against citrullinated peptides (ACPA). RA is influenced by genetic factors, specifically, there is a strong genetic association with the shared epitope (SE), a five amino acid sequence motif in positions 70–74 of HLA-DRβ1 chains encoded by HLA-DRB1 alleles: QKRAA, QRRAA and RRRAA.The present study aims to analyze whether SE-peptides (SE-p) can be a target of the autoimmune response in RA.Objectives:To analyze the presence of AAb against the unmodified (Un) SE-p, citrullinated (Cit) SE-p and carbamylated (Car) SE-p.Methods:Sera from consecutive 117 RA patients and 21 psoriasic arthritis (PsA) from our outpatient clinic were collected by venopunture. Also 138 sera from blood donors were obtained as healthy controls (HC). All participants signed the informed consent.We perfomed a homemade ELISA test using a sequence of 15 aminoacid peptides from positions 65-79 of HLA-DRB1 containing the 3 different SE sequences, in the Un, Cit and Car SE-p, synthesized in a linear and cycled form. We established a 90% of specificity using a ROC curve obtained from HC and PsA for each ELISA test.HLA-DRB1 polymorphism was performed using a HLA-DRB1 sequence specific oligonucleotide typing kit (Lifecodes) in 95 RA and in 15 PsA.ACPA and RF were determined with commercial assays (Inova Diagnostics and Binding Site, respectively).Results:The overall sensitivity of the different SE-p AAb tests ranged from 5.1-21.4%.RRRAA SE polymorphism was associated with AAb against cycled CitCitCitAA SE-p (p=0.025), QKRAA polymorphism was almost significantly associated with AAb against cycled QKCitAA SE-p (p=0.067), whereas there was no association between QRRAA polymorphism and AAb against cycled QCitCitAA SE-p (p=0.690). On the other hand, there was no association between SE polymorphisms and AAb to any other peptide used in the ELISA test.Significant differences were observed in the presence of AAb against lineal RRRAA, lineal CitCitCitAA and cycled CitCitCitAA SE-p when comparing RA vs. HC patients (p=0.022, 0.044, 0.022, respectively). Moreover, there also were significant differences in the presence of AAb against cycled CitCitCitAA SE-p between RA and PsA patients (sensitivity 21.4%, specificity 100%; p=0.014).It must be highlighted that cycled CitCitCitAA SE-p AAb were detected in 20.0% of RA patient sera that were negative for RF and ACPA.There was no association between RF or ACPA with the presence of any SE-p AAb.Conclusion:RA patients have autoantibodies against the Shared Epitope (SE). The cycled CitCitCitAA SE peptide (SE-p) shows the best performance among all the peptides tested and could identify patients seronegative for ACPA and RF, both analyzed by commercial assays.Additional studies must be performed to verify the diagnostic and utility of these new autoantibodies against SE-p in RA.Acknowledgements:This work was granted by the 2018 call of the “Fundación Española de Reumatologia” and the 2017 call grant “Fundació Parc Taulí”.Disclosure of Interests:None declared

POS1429 ORAL DYSBIOSIS REFLECTS THE IMMUNOLOGICAL ALTERATION OF RA REGARDING TO HLA DRB1*SE, ACPA AND CIGARETTE SMOKING: NAGASAKI ISLAND STUDY

Background:Anti-citrullinated protein antibody (ACPA) production is observed in several organs even prior to the onset of rheumatoid arthritis (RA), and oral mucosa is considered to be one of the important tissues. Saliva is considered to reflect the oral microbiota(oralMB) including periodontal disease. A gene-environment interaction between cigarette smoking and shared epitope genes in HLA-DRB1*shared epitope (SE) provides a high risk of ACPA-positive RA. However, the interaction of HLA-DRB1*SE, ACPA, cigarette smoking and oralMB of RA patients remains to be elucidated.Objectives:We investigated that the difference of oralMB among RA patients and healthy subjects(HS) regarding to ACPA, HLA-DRB1*SE and cigarette smoking.Methods:The Nagasaki Island Study, which had started in 2014 collaborating with Goto City, Nagasaki Prefecture, Japan, is intended for research of the preclinical stage of RA, including ACPA, HLA genotype screening, oralMB and lifestyle habit. Both of blood and salivary samples were obtained from 1422 subjects out of 4276 participants in this study from 2016 to 2018. ACPA positivity was 1.7 % in total 4276 subjects. At this point, we selected 291 subjects, who were ACPA positive non-RA HS(n=22) and patients with RA (n=33, 11 subjects were ACPA positive and 22 ACPA negative, respectively) as the case, age and gender matched ACPA negative non-RA HS (n=236) as the control. In RA subjects, current smoker was n=1(3.0%) and ever smoker was n=8(24.2%). In HS, current smoker was n=29(11.2%) and ever smoker was n=55(21.3%). ACPA was measured by ELISA, and HLA genotyping was quantified by next-generation sequencing (Ref.1). The operational taxonomic unit (OTU) analysis using 16S rRNA gene sequencing were performed. The richness of microbial diversity within subject (α-diversity) was scaled via Shannon entropy. The dissimilarity between microbial community composition was calculated using Bray-Curtis distance as a scale, and differences between groups (β-diversity) were tested by permutational multivariate analysis of variance (PERMANOVA). In addition, UniFrac distance calculated in consideration of the distance on the phylogenetic tree were performed.Results:Median age 71 y.o., % Female 58.4 %. Among RA and non-RA subjects, not α-diversity but β-diversity was statistically smaller significantly in RA (p=0.022). In the HS, there was no decrease in α-diversity between the ACPA-positive and HLA-DRB1*SE-positive groups, but in the ACPA-positive group, there was a decrease in α-diversity in the HLA-DRB1*SE-positive group. When we compared α-diversity stratified by the presence or absence of three factors (RA, ACPA, and HLA-DRB1*SE), the RA group with ACPA and HLA-DRB1*SE positive tended to have the lowest diversity (Figure 1 lower right). RA subjects, presence of HLA-DRB1*SE did not show the difference but the tendency of lower α-diversity (p=0.29).Conclusion:HS with ACPA-positive HLA-DRB1*SE tended to show lower α-diversity than ACPA-positive HS and HLA-DRB1*SE positive HS. Furthermore, RA subjects with ACPA-positive HLA-DRB1*SE showed lower α-diversity than HS with ACPA-positive HLA-DRB1*SE.Our study suggested that the oral dysbiosis may reflect the immunological status of patients with RA. Because of the small number of ACPA-positive patients, stratification by smoking history was difficult. Further examination is needed to clarify the gene-environment interaction and microbiome.References:[1]Kawaguchi S, et al. Methods Mol Biol 2018;1802: 22.Disclosure of Interests:None declared

Influence of the shared epitope on the elicitation of experimental autoimmune arthritis biomarkers

Our previous studies have shown that inoculation of the oral cavity of “humanized” B6.DR1/4 mice with the periodontal pathogen Porphyromonas gingivalis results in an increase in the percentage of circulating Th17 cells, loss of bone and an exacerbation of experimental autoimmune arthritis. The aim of this study was to assess the role played by the human HLA-DRβ molecule containing the shared epitope supplied as a transgene to I-A˚ (murine class II null) C57BL/6 (B6) mice in driving these findings. We compared various immune response parameters as well as alveolar and peri-articular bone loss between humanized B6.DR1 (or B6.DR4) mice and their WT (B6) counterparts. We found that the presence of the shared epitope in the context of inoculation with P. gingivalis enhanced the percentage of Th17 cells generated, dramatically enhanced bone loss and importantly allowed for the generation of CCP2⁺ ACPAs that are not found in C57BL/6 or DBA/1 arthritic mouse serum. Due to the exceedingly complex nature of environmental factors impacting on genetic elements, it has been difficult to unravel mechanisms that drive autoimmune arthritis in susceptible individuals. The findings in this study may provide one small piece of this puzzle that can help us to better understand part of this complexity.

Altered Antibody Response to Epstein-Barr Virus in Patients With Rheumatoid Arthritis and Healthy Subjects Predisposed to the Disease. A Twin Study

Objectives: To study Epstein-Barr virus (EBV) antibody patterns in twin individuals with rheumatoid arthritis (RA) and their healthy co-twins, and to determine the heritability of antibody responses against the EBV encoded EBNA1 protein.Methods: Isotypes of EBNA1 antibodies were measured in 137 RA affected- and 150 healthy twin pairs. We estimated the effect of RA and RA predisposition, anti-citrullinated antibodies (ACPA), IgM rheumatoid factor (RF), the shared epitope (SE) and the PTPN22-T allele (PTPN22) on the level of EBNA1 antibodies. We also determined the heritability of EBNA1 antibody levels.Results: IgA-EBNA1 antibody levels were increased in twins from RA discordant twin pairs irrespective of RA, ACPA or IgM-RF status. The IgG-EBNA1 antibody level was elevated in healthy co-twins from RA discordant twin pairs but not in RA affected twins. The IgM-EBNA1 antibody level was elevated in both RA twins and their healthy co-twins. The effect of RA on the IgA-EBNA1 antibody level was reversed when SE was present and with no effect of PTPN22. The heritability of IgA-, IgG- and IgM-EBNA1 antibody level was 40.6, 65.5, and 54.3%, with no effect of environment shared by the twins.Conclusion: EBNA1 antibody levels are distinctively different between patients with RA and healthy subjects but also between relatives of RA strongly predisposed to RA and healthy subjects. The high level of IgA EBNA1 antibodies associated with RA and a family predisposition to RA is attributable to both genetics incl. the shared epitope and environmental variation.

Contribution of the Human Microbiome and Proteus mirabilis to Onset and Progression of Rheumatoid Arthritis: Potential for Targeted Therapy

The human microbiome has been shown to play a role in the regulation of human health, behavior, and disease. Data suggests that microorganisms that co-evolved within humans have an enhanced ability to prevent the development of a large spectrum of immune-related disorders but may also lead to the onset of conditions when homeostasis is disrupted. In many conditions, a link between dysbiosis (microbial imbalance or microbiome upset) has been identified and associated with immune conditions such as rheumatoid arthritis (RA). This review provides insight into how an individual’s unique microbiome, combined with a genetic predisposition and environmental factors may lead to the onset and progression of RA. While research efforts have been largely focused on Porphyromonas gingivalis in the generation of citrullinated products as a trigger in the onset and progression of RA, recent research efforts have also indicated that Proteus mirabilis may play a key role in the development of anti-citrullinated antibodies through shared epitope sequences IRRET and ESRRAL. Thus, this review also highlights how targeting dysbiosis with alternative approaches may help to reduce microbial resistance as well as potentially improve outcomes. Further investigation is needed to see if potential future treatments for RA could benefit from personalized medicine based on an individual’s unique microbiome