Anti-modified Protein Antibodies Research Articles

Presence of anti-modified protein antibodies in idiopathic pulmonary fibrosis.

Studies of autoimmunity and anti-citrullinated protein antibodies (ACPA) in idiopathic pulmonary fibrosis (IPF) have been confined to investigations of anti-cyclic citrullinated peptide (anti-CCP) antibodies which utilize synthetic peptides as surrogate markers for in vivo citrullinated antigens. We studied immune activation by analysing the prevalence of in vivo anti-modified protein antibodies (AMPA) in IPF. We included patients with incident and prevalent IPF (N = 120), sex and smoking-matched healthy controls (HC) (N = 120) and patients with RA (N = 104). Serum (median time: 11 months [Q1-Q3: 1-28 months] from diagnosis) was analysed for presence of antibodies towards native and posttranslational modified (citrullinated [Cit, N = 25]; acetylated [Acet, N = 4] and homocitrullinated [Carb, N = 1]) peptides derived from tenascin (TNC, N = 9), fibrinogen (Fib, N = 11), filaggrin (Fil, N = 5), histone (N = 8), cathelicidin (LL37, N = 4) and vimentin (N = 5) using a custom-made peptide microarray. AMPA were more frequent and in increased levels in IPF than in HC (44% vs. 27%, p < 0.01), but less than in RA (44% vs. 79%, p < 0.01). We specifically observed AMPA in IPF towards certain citrullinated, acetylated and carbamylated peptides versus HC: tenascin (Cit(2033) -TNC2025-2040 ; Cit(2197) -TNC2177-2200 ; Cit(2198) -TNC2177-2200 ), fibrinogen (Cit(38,42) -Fibα36-50 ; Cit(72) -Fibβ60-74 ) and filaggrin (Acet-Fil307-324 , Carb-Fil307-324 ). No differences in survival (p = 0.13) or disease progression (p = 0.19) between individuals with or without AMPA was observed in IPF. However, patients with incident IPF had better survival if AMPA were present (p = 0.009). A significant proportion of IPF patients present with specific AMPA in serum. Our results suggest autoimmunity as a possible characteristic for a subgroup of IPF that may affect disease outcome.

Geo-epidemiology of autoantibodies in rheumatoid arthritis: comparison between four ethnically diverse populations

BackgroundRheumatoid arthritis (RA) occurs across the globe in different ethnic populations. Most RA patients harbor anti-modified protein antibodies (AMPA); however, it is unclear whether differences exist in autoantibody responses at different geographic locations and between different ethnic groups, which could provide new clues regarding factors underlying autoantibody development. We therefore investigated AMPA prevalence and association with HLA DRB1 alleles and smoking in four ethnically diverse populations on four different continents.MethodsAnti-carbamylated (anti-CarP), anti-malondialdehyde acetaldehyde (anti-MAA), and anti-acetylated protein antibodies (anti-AcVim) IgG were determined in anti-citrullinated protein antibody-positive Dutch (NL, n = 103), Japanese (JP, n = 174), First Nations Peoples in Canada (FN, n = 100), and black South African (SA, n = 67) RA patients. Ethnicity-matched local healthy controls were used to calculate cut-offs. Risk factors associated with AMPA seropositivity in each cohort were identified using logistic regression.ResultsMedian AMPA levels were higher in First Nations Peoples in Canada and especially South African patients, as reflected by percentage seropositivity: NL, JP, FN, and SA: anti-CarP: 47%, 43%, 58%, and 76% (p < 0.001); anti-MAA: 29%, 22%, 29%, and 53% (p < 0.001); and anti-AcVim: 20%, 17%, 38%, and 28% (p < 0.001). Total IgG levels also differed markedly, and when autoantibody levels were normalized to total IgG, differences between cohorts became less pronounced. Although there were some associations with AMPA and HLA risk alleles and smoking, none was consistent across all four cohorts.ConclusionsAMPA against various post-translational modifications could consistently be detected on different continents across ethnically diverse RA populations. Differences in AMPA levels corresponded to differences in total serum IgG levels. This suggests that, despite differences in risk factors, a common pathway may be involved in AMPA development across geographic locations and ethnicities.

Anticitrullinated antibodies recognize rheumatoid arthritis associated T-cell epitopes modified by bacterial L-asparaginase.

Citrullinated proteins and anti-citrullinated protein antibodies (ACPAs) play an important role in the pathogenesis of rheumatoid arthritis (RA). It has been suggested that during inflammation or dysbiosis, bacteria could initiate production of ACPAs. Most patients with RA are seropositive for ACPAs, but these antibodies have overlapping reactivity to different posttranslational modifications (PTMs). For initiation and development of RA, T lymphocytes and T cell epitopes are still required. In this study, we evaluated the ability of bacterial L-asparaginase to modify RA-related T cell epitopes within type II collagen (CII259-273 and CII311-325), as well as whether these modified epitopes are recognized by ACPAs from RA patients. We included 12 patients with early RA and 11 healthy subjects selected according to predefined specific criteria. LC-MS/MS analyses revealed that the bacterial L-asparaginase can modify investigated T cell epitopes. ELISA tests showed cross-reactivity of ACPA positive sera from early RA patients towards the enzymatically modified immunodominant T cell epitopes within type II collagen (CII), but not to the modified irrelevant peptides. These data suggest that the cross-reactive ACPAs recognize the "carbonyl-Gly-Pro" motif in CII. Moreover, the T cell recognition of the modified major immunodominant T cell epitope Gal264-CII259-273 was not affected. This epitope was still able to activate autoreactive T cells from early RA patients. It is likely that such modifications are the missing link between the T cell priming and the development of anti-modified protein antibodies (AMPAs). Our results provide additional information on the etiology and pathogenesis of RA.

Autoantibodies targeting malondialdehyde-modifications in rheumatoid arthritis regulate osteoclasts via inducing glycolysis and lipid biosynthesis

Proteins subjected to post-translational modifications, such as citrullination, carbamylation, acetylation or malondialdehyde (MDA)-modification are targeted by autoantibodies in seropositive rheumatoid arthritis (RA). Epidemiological and experimental studies have both suggested the pathogenicity of such humoral autoimmunity, however, molecular mechanisms triggered by anti-modified protein antibodies have remained to be identified.Here we describe in detail the pathways induced by anti-MDA modified protein antibodies that were obtained from synovial B cells of RA patients and that possessed robust osteoclast stimulatory potential and induced bone erosion in vivo. Anti-MDA antibodies boosted glycolysis in developing osteoclasts via an FcγRI, HIF-1α and MYC-dependent mechanism and subsequently increased oxidative phosphorylation. Osteoclast development required robust phosphoglyceride and triacylglyceride biosynthesis, which was also enhanced by anti-MDA by modulating citrate production and expression of the glycerol-3-phosphate dehydrogenase 1 (GPD1) and glycerol-3-phosphate acyltransferase 2 (GPAT2) genes. In summary, we described novel metabolic pathways instrumental for osteoclast differentiation, which were targeted by anti-MDA antibodies, accelerating bone erosion, a central component of RA pathogenesis.

POS0531 ABATACEPT DELAYS THE DEVELOPMENT OF RA– CLINICAL RESULTS AFTER 18 MONTHS FROM THE RANDOMIZED, PLACEBO-CONTROLLED ARIAA STUDY IN RA-AT RISK PATIENTS

BackgroundThe development of RA is described by a preclinical phase of autoimmunity, that precedes clinical disease. This autoimmune phase is characterized by the presence of anti-modified protein antibodies that recognize citrullinated proteins (ACPA). A subset of individuals with ACPA develops RA, i.e. those with imaging signs of subclinical inflammation in the joints. As T cell mediated B cell activation is a key step in developing autoimmunity and RA, interventions that target this process may be useful for preventing the onset of RA. In this context, abatacept seems being an attractive therapeutic tool as it interrupts the activation of T cells and has a favourable safety profile in the treatment of RA.ObjectivesTo test whether treatment of abatacept, as compared to placebo, delays the onset of RA in ACPA positive individuals with a high risk to develop RA.MethodsARIAA is an international, randomized double-blinded placebo-controlled multi-center study in RA-at risk individuals, being ACPA positive and showing MRI signs of inflammation. The study was composed of a 6 months treatment phase with either abatacept s.c. 125 mg weekly or placebo and a 12 months observation phase with no treatment. Primary endpoint was the improvement of MRI inflammation after 6 months, secondary endpoints were the progression to RA after 6 and 18 months. The primary analysis was done on the ITT population and missing values were classified as treatment failures.ResultsBetween November 2014 and December 2019 139 RA-at risk individuals were included into ARIAA by 14 study sites (11 in Germany, 1 in the Czech Republic and 2 in Spain). Of them, 100 patients were randomized to receive either abatacept or placebo. Two patients were excluded and 98 patients could be evaluated for efficacy and safety. The primary endpoint was met: 61% of abatacept and 31% of placebo treated individuals (p=0.0043) improved in MRI inflammation. Furthermore, only 4 patients (8.2%) in the abatacept group but 17 patients in the placebo group (34.7%) progressed to RA after 6 months (p= 0.0025). Even 1 year after cessation of treatment (18 months after inclusion) the number of patients progressing to RA was lower in the abatacept group (35%) than in the placebo group (57%; p=0.0421). With respect to safety, 12 serious adverse events (each one gastritis, cellulitis, pneumonia, tendinitis calcificans, rotator cuff syndrome, cholelithiasis, peripheral artery disease, idiopathic pain syndrome, prostate cancer, penile neoplasm; trabeculectomy, cataract surgery) were reported, with only one (pneumonia) being considered to be related to treatment.ConclusionAbatacept significantly reduces subclinical joint inflammation and delays the development of RA in at-risk individuals.Table 1.ABAPBOAllN494998Females, N (%)31 (63.3)39 (79.6)70 (71.4)Age (ys); mean (±SD)51.3 (±10.8)48.5 (±12.6)49.9 (±11.7)SJC (N); mean (±SD)000TJC (N); mean (±SD)3.06 (± 3.75)3.51 (± 4.42)3.29 (±4.08)VAS Pain (mm) mean (±SD)42.2 (± 27.1)42.8 (± 33.2)42.5 (± 30.2)VAS PG (mm) mean (±SD42.2 (±28.7)43.0 (± 33.8)42.6 (± 31.2)MRI improvement, N (%)30 (61.2)15 (30.6)45 (45.9)RA at 6 months, N (%)4 (8.2)17 (34.7)21 (21.4)RA at 18 months, N (%)17 (34.7)28 (57.1)45 (45.9)AcknowledgementsThe study was supported by BMS according to the items outlined in the IIS contract and the IMI funded project RTCure.Disclosure of InterestsJürgen Rech Consultant of: Abbvie, Biogen, BMS, Chugai, GSK, Lilly, MSD; Novartis, Roche, Sanofi, Sobi, UCB, Consultancy: Biogen, BMS, Chugai, GSK, Lilly, MSD, Novartis, Roche, Sanofi, Sobi, UCB, Grant/research support from: Sobi, Novartis, Arnd Kleyer Consultant of: BMS, Pfizer, Sanofi, Abbvie, Janssen, Medac, Novartis, Lilly Deutschland GmbH, Gilead, Amgen, Grant/research support from: Novartis, Lilly Deutschland GmbH, Mikkel Østergaard: None declared, Melanie Hagen: None declared, Larissa Valor: None declared, Koray Tascilar: None declared, Gerhard Krönke: None declared, Verena Schönau: None declared, Stefan Kleinert: None declared, Xenofon Baraliakos: None declared, Juergen Braun: None declared, Martin Fleck: None declared, Andrea Rubbert-Roth: None declared, Frank Behrens: None declared, Martin Feuchtenberger: None declared, Michael Zaenker: None declared, David M Kofler: None declared, Reinhard Voll: None declared, Cornelia Glaser: None declared, Axel Hueber: None declared, Eugen Feist: None declared, Gerd Rüdiger Burmester: None declared, Kirsten Karberg: None declared, Johannes Strunk: None declared, Juan de Dios Cañete: None declared, Ladislav Šenolt: None declared, Esperanza Naredo: None declared, Georg Schett: None declared.

Rheumatoid Factor and Anti-Modified Protein Antibody Reactivities Converge on IgG Epitopes.

Rheumatoid arthritis (RA) patients often develop rheumatoid factors (RFs), antibodies that bind IgG Fc, and anti-modified protein antibodies (AMPAs), multireactive autoantibodies that commonly bind citrullinated, homocitrullinated, and acetylated antigens. Recently, antibodies that bind citrulline-containing IgG epitopes were discovered in RA, suggesting that additional undiscovered IgG epitopes could exist and that IgG could be a shared antigen for RFs and AMPAs. This study was undertaken to reveal new IgG epitopes in rheumatic disease and to determine if multireactive AMPAs bind IgG. Using sera from patients with RA, systemic lupus erythematosus, Sjögren's disease (SjD), or spondyloarthropathy, IgG binding to native, citrulline-containing, and homocitrulline-containing linear epitopes of the IgG constant region was evaluated by peptide array, with highly bound epitopes further evaluated by enzyme-linked immunosorbent assay (ELISA). Binding of monoclonal AMPAs to IgG-derived peptides and IgG Fc was also evaluated by ELISA. Seropositive RA sera showed high IgG binding to multiple citrulline- and homocitrulline-containing IgG-derived peptides, whereas anti-SSA+ sera from SjD patients showed consistent binding to a single linear native epitope of IgG in the hinge region. Monoclonal AMPAs bound citrulline- and homocitrulline-containing IgG peptides and modified IgG Fc. The repertoire of epitopes bound by AMPAs includes modified IgG epitopes, positioning IgG as a common antigen that connects the otherwise divergent reactivities of RFs and AMPAs.

Microstructural Bone Changes Are Associated With Broad‐Spectrum Autoimmunity and Predict the Onset of Rheumatoid Arthritis

To assess if microstructural bone lesions in individuals at risk of developing rheumatoid arthritis (RA) are related to the spectrum of anti-modified protein antibodies (AMPAs) and affect the risk of developing RA. Cortical microchannels as well as cortical and trabecular bone mineral density (BMD) volumes (expressed as mg hydroxyapatite/cm3 ) were analyzed by high-resolution peripheral quantitative computed tomography of the hand joints of individuals at risk of RA. AMPA response was profiled, including reactivities against citrullinated proteins (vimentin, enolase, and fibrinogen) as well as carbamylated and acetylated vimentin. All subjects were followed up for the development of RA. Subjects at risk of developing RA (n=75) who had broad-spectrum AMPAs (6-8 reactivities) had significantly more severe microstructural changes, including a higher mean ± SD number of cortical microchannels per joint (95 ± 3) and lower total volumetric BMD (vBMD; 265 ± 45), trabecular vBMD (176 ± 42), and cortical vBMD (585 ± 138), than those with moderate AMPA reactivity (3-5 reactivities) (number of cortical microchannels, 79 ± 30; total vBMD, 293 ± 33; trabecular vBMD, 195 ± 32; and cortical vBMD, 627 ± 91) and those with narrow AMPA reactivity (1-2 reactivities) (number of cortical microchannels, 47 ± 20; total vBMD, 311 ± 34; trabecular vBMD, 211 ± 30; and cortical vBMD, 674 ± 56). Progressors to RA had significantly higher numbers of cortical microchannels (103 ± 30 versus 71 ± 35) and lower bone volume (258 ± 37 versus 295 ± 34) compared to nonprogressors. Furthermore, rate of progression to RA was high in subjects with broad AMPA reactivity (48%) versus those with medium AMPA reactivity (26%) or narrow AMPA reactivity (0%), as well as in those with a high number of cortical microchannels (44%) versus those with a low number of cortical microchannels (10%). Microstructural changes in individuals at risk of RA are associated with broad-spectrum autoimmunity and predict the onset of RA. These data support the concept of structural priming of joints by autoimmunity before the onset of the inflammatory phase of the disease.

Impact of Posttranslational Modification in Pathogenesis of Rheumatoid Arthritis: Focusing on Citrullination, Carbamylation, and Acetylation.

Rheumatoid arthritis (RA) is caused by prolonged periodic interactions between genetic, environmental, and immunologic factors. Posttranslational modifications (PTMs) such as citrullination, carbamylation, and acetylation are correlated with the pathogenesis of RA. PTM and cell death mechanisms such as apoptosis, autophagy, NETosis, leukotoxic hypercitrullination (LTH), and necrosis are related to each other and induce autoantigenicity. Certain microbial infections, such as those caused by Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Prevotella copri, can induce autoantigens in RA. Anti-modified protein antibodies (AMPA) containing anti-citrullinated protein/peptide antibodies (ACPAs), anti-carbamylated protein (anti-CarP) antibodies, and anti-acetylated protein antibodies (AAPAs) play a role in pathogenesis as well as in prediction, diagnosis, and prognosis. Interestingly, smoking is correlated with both PTMs and AMPAs in the development of RA. However, there is lack of evidence that smoking induces the generation of AMPAs.

Cross-reactivity of IgM anti-modified protein antibodies in rheumatoid arthritis despite limited mutational load

BackgroundAnti-modified protein antibodies (AMPA) targeting citrullinated, acetylated and/or carbamylated self-antigens are hallmarks of rheumatoid arthritis (RA). Although AMPA-IgG cross-reactivity to multiple post-translational modifications (PTMs) is evident, it is unknown whether the first responding B cells, expressing IgM, display similar characteristics or if cross-reactivity is crucially dependent on somatic hypermutation (SHM). We now studied the reactivity of (germline) AMPA-IgM to further understand the breach of B cell tolerance and to identify if cross-reactivity depends on extensive SHM. Moreover, we investigated whether AMPA-IgM can efficiently recruit immune effector mechanisms.MethodsPolyclonal AMPA-IgM were isolated from RA patients and assessed for cross-reactivity towards PTM antigens. AMPA-IgM B cell receptor sequences were obtained by single cell isolation using antigen-specific tetramers. Subsequently, pentameric monoclonal AMPA-IgM, their germline counterparts and monomeric IgG variants were generated. The antibodies were analysed on a panel of PTM antigens and tested for complement activation.ResultsPentameric monoclonal and polyclonal AMPA-IgM displayed cross-reactivity to multiple antigens and different PTMs. PTM antigen recognition was still present, although reduced, after reverting the IgM into germline. Valency of AMPA-IgM was crucial for antigen recognition as PTM-reactivity significantly decreased when AMPA-IgM were expressed as IgG. Furthermore, AMPA-IgM was 15- to 30-fold more potent in complement-activation compared to AMPA-IgG.ConclusionsWe provide first evidence that AMPA-IgM are cross-reactive towards different PTMs, indicating that PTM (cross-)reactivity is not confined to IgG and does not necessarily depend on extensive somatic hypermutation. Moreover, our data indicate that a diverse set of PTM antigens could be involved in the initial tolerance breach in RA and suggest that AMPA-IgM can induce complement-activation and thereby inflammation.

Antigen-driven autoantibody production in lungs of interstitial lung disease with autoimmune disease

Interstitial lung disease (ILD) sometimes becomes a life-threatening complication of systemic autoimmune diseases; however, little is known about the immune response in lung lesions. We aimed to investigate humoural immunity in ILD associated with rheumatoid arthritis (RA), sjögren's syndrome (SjS), and mixed connective tissue disease (MCTD), using bronchoalveolar fluid (BALF) and serum samples from 15 patients with autoimmune disease associated-ILD. We first showed that BALF contained higher titers of disease-related autoantibodies than serum, suggesting the possibility of autoantibody production in lungs. Next, we produced 326 monoclonal antibodies from antibody-secreting cells in BALF, and the reactivity and their revertants, in which somatic hypermutations were reverted to germline, were analyzed. Among 123 antibodies from RA-ILD, 16 disease-related antibodies (anti-modified protein antibodies and rheumatoid factors) were identified, of which one antibody had both properties. The revertant antibodies changed their target modification in a complicated manner, suggesting that the antibodies were selected against various modifications in lungs. Among 146 antibodies from SjS-ILD and/or MCTD-ILD, seven anti-SSA/Ro60 antibodies and 15 anti-RNP antibodies were identified. Some of the anti-RNP antibodies recognized multiple RNP constituent proteins simultaneously, indicating that epitope spreading may progress in lungs. Our results revealed the existence of an active autoimmunity in the lungs of autoimmune disease associated-ILD.

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

The impact of autoantibodies against citrullinated, carbamylated, and acetylated peptides on radiographic progression in patients with new-onset rheumatoid arthritis: an observational cohort study.

A range of anti-modified protein antibodies (AMPAs) are associated with rheumatoid arthritis. We aimed to assess the relationship between AMPA profiles and radiographic progression in patients with new-onset rheumatoid arthritis. In this cohort study, we obtained samples and data from the Scottish Early Rheumatoid Arthritis (SERA) inception cohort and biobank, which recruited patients with new-onset rheumatoid arthritis or undifferentiated arthritis who had at least one swollen joint from 20 hospitals across Scotland. AMPAs in plasma samples were measured by ELISAs at baseline. Paired radiographs of the hands and feet were taken at baseline and at 1 year and were scored with the Sharp-van der Heijde (SvH) method. We calculated differences in radiographic progression using estimated marginal mean changes between baseline and 1 year, with the baseline values of radiographic variables, rheumatoid factor, sex, age at recruitment, symptom duration, and Disease Activity Score 28 with C-reactive protein included as covariates. Between March 1, 2011, and April, 30, 2015, 1073 patients were recruited to the SERA study. 362 patients with rheumatoid arthritis were included in our study and had their AMPA profiles determined. Patients were grouped into four main autoantibody profiles by reactivities to post-translational modifications: single positivity for anti-citrullinated peptide antibodies (ACPAs; 73 [20%]); double positivity for ACPAs and anti-acetylated peptide antibodies (AAPAs; 45 [12%]); triple positivity for ACPAs, AAPAs, and anti-carbamylated peptide antibodies (151 [42%]); and AMPA negativity (74 [20%]). 19 (5%) patients were in one of the minor autoantibody groups. Of the 233 patients with both antibody data and radiographs of sufficient quality, triple-positive patients had more radiographic progression between baseline and 12 months (estimated mean change in total SvH score 1·8, 95% CI 0·9-2·6, SE 0·4) than did single-positive patients (0·5, 0·1-1·0, 0·2; estimated mean difference in the total change in SvH score 1·2, 95% CI 0·1-2·4, SE 0·5). There was no difference in radiographic progression between single positive patients and AMPA negative patients (estimated mean change in total SvH score 0·7, 95% CI 0·1-1·4, SE 0·3; estimated mean difference in the total change in SvH score -0·2, 95% CI -1·1 to 0·7, SE 0·4). This study suggests that the optimal prediction of future rates of radiographic progression in patients with rheumatoid arthritis will require an assessment of autoantibodies against multiple post-translationally modified proteins or peptides. The EU FP7 HEALTH programme, the Scottish Translational Medicine Research Collaboration, and the Chief Scientist Office Scotland.

Causal risk and protective factors in rheumatoid arthritis: A genetic update

The characterization of risk and protective factors in complex diseases such as rheumatoid arthritis (RA) has evolved from epidemiological studies, which test association, to the use of Mendelian randomization approaches, which test direct relationships. Indeed, direct associations with the mucosal origin of RA are retrieved with periodontal disease (Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans predominantly), interstitial lung involvement, tobacco smoking and air pollutants. Next, factors directly associated with an acquired immune response include genetic factors (HLA DRB1, PTPN22), capacity to produce anti-modified protein antibodies (AMPA), and relatives with a history of autoimmune diseases. Finally, factors can be also classified according to their direct capacity to interfere with the IL-6/CRP/sIL-IL6R proinflammatory pathway as risk factor (body fat, cardiometabolic factors, type 2 diabetes, depressive syndrome) or either as protective factors by controlling of sIL-6R levels (higher education level, and intelligence). Although some co-founders have been characterized (e.g. vitamin D, physical activity, cancer) the direct association with sex-discrepancy, pregnancy, and infections among other factors remains to be better explored.

SAT0585 GEO-EPIDEMIOLOGY OF AUTOANTIBODIES IN RA: DIFFERENT PREVALENCES IN FOUR ETHNICALLY DIVERSE RA POPULATIONS

Background:Rheumatoid arthritis (RA) has been described in virtually every ethnic population. Most RA patients harbor anti-modified protein antibodies (AMPAs), including anti-citrullinated protein (ACPA), anti-carbamylated protein (anti-CarP), anti-malondialdehyde acetaldehyde (anti-MAA), and anti-acetylated protein antibodies (AAPA). However, it is unclear whether differences exist in the AMPA response between different ethnic groups. Such differences could provide new clues to genetic and environmental factors contributing to autoantibody development.Objectives:To investigate the prevalence of different AMPA in four ethnically diverse RA populations, and their association with smoking.Methods:Enzyme-linked immunosorbent assays were used to measure anti-CarP IgG, anti-MAA IgG (both in-house), and anti-acetylated vimentin IgG (Orgentec) in ACPA-positive sera of Dutch (NL, n=103), Japanese (JP, n=174), Canadian First Nations People (FN, n=100), and black South Africans (SA, n=67) fulfilling the 1987 ACR classification criteria for RA. Ethnicity-matched local healthy controls were used to calculate cohort-specific cut-offs. Logistic regression was used to identify whether ever-smoking was associated with AMPA seropositivity in each cohort, corrected for age, gender, and disease duration. Random-effects meta-analysis was used to pool the resulting odds ratios (OR).Results:For all three AMPAs, median levels were higher in FN and especially SA than NL and JP patients (Figure 1). The median autoantibody levels in arbitrary units (in % of patients positive) for NL, JP, FN and SA RA patients were: anti-CarP IgG: 1157 (47%), 994 (43%), 1642 (58%) and 2336 (76%) (p&lt;0.001); anti-MAA IgG: 131 (29%), 179 (22%), 251 (29%) and 257 (53%) (p&lt;0.001); AAPA: 133 (20%), 136 (17%), 153 (38%) and 316 (28%) (p&lt;0.001). Prevalence, meaning positivity, also differed significantly between cohorts for all AMPAs (p&lt;0.001).There were also marked differences in total IgG levels in mean (SD) g/L: 13 (4) for NL, 17 (6) for JP, 18 (6) for FN, and 25 (8) for SA (p&lt;0.001). When the autoantibody levels were normalized to total IgG, the differences in became less pronounced between cohorts (Figure 2). The median arbitrary units per g/L Total IgG for NL, JP, FN and SA RA patients were: anti-CarP IgG: 54, 25, 53, and 79; anti-MAA IgG: 6, 5, 8, and 9; and AAPA: 2, 2, 2, and 3, suggesting that autoantibody level differences may partly correspond to cohort-specific differences in total IgG, although the overall trend of higher levels in SA persisted. There was no association between smoking and anti-CarP or anti-MAA positivity, with pooled OR (95% CI) of 1.31 (0.79-2.18) and 0.85 (0.46-1.56), respectively. However, smoking was positively and consistently associated with AAPA positivity in each cohort: pooled OR (95% CI) of 2.01 (1.06-3.81).Conclusion:In these ACPA-positive ethnically diverse RA populations, levels and prevalence of various AMPAs differ, suggesting that ethnic background and environment may influence the development of the autoantibody response in RA. Despite these differences, our results imply smoking as a consistent risk factor for AAPA across different ethnic backgrounds.Disclosure of Interests:Emma C. de Moel: None declared, Veerle Derksen: None declared, Leendert A Trouw: None declared, Chikashi Terao: None declared, Mohammed Tikly: None declared, Hani El-Gabalawy: None declared, Holger Bang Grant/research support from: Employee of Orgentec Diagnostika, Thomas Huizinga Grant/research support from: Ablynx, Bristol-Myers Squibb, Roche, Sanofi, Consultant of: Ablynx, Bristol-Myers Squibb, Roche, Sanofi, Rene Toes: None declared, Diane van der Woude: None declared

Autoantibodies and B Cells: The ABC of rheumatoid arthritis pathophysiology.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by joint inflammation. In the last few decades, new insights into RA‐specific autoantibodies and B cells have greatly expanded our understanding of the disease. The best‐known autoantibodies in RA—rheumatoid factor (RF) and anti‐citrullinated protein antibodies (ACPA)—are present long before disease onset, and both responses show signs of maturation around the time of the first manifestation of arthritis. A very intriguing characteristic of ACPA is their remarkably high abundance of variable domain glycans. Since these glycans may convey an important selection advantage of citrulline‐reactive B cells, they may be the key to understanding the evolution of the autoimmune response. Recently discovered autoantibodies targeting other posttranslational modifications, such as anti‐carbamylated and anti‐acetylated protein antibodies, appear to be closely related to ACPA, which makes it possible to unite them under the term of anti‐modified protein antibodies (AMPA). Despite the many insights gained about these autoantibodies, it is unclear whether they are pathogenic or play a causal role in disease development. Autoreactive B cells from which the autoantibodies originate have also received attention as perhaps more likely disease culprits. The development of autoreactive B cells in RA largely depends on the interaction with T cells in which HLA “shared epitope” and HLA DERAA may play an important role. Recent technological advances made it possible to identify and characterize citrulline‐reactive B cells and acquire ACPA monoclonal antibodies, which are providing valuable insights and help to understand the nature of the autoimmune response underlying RA. In this review, we summarize what is currently known about the role of autoantibodies and autoreactive B cells in RA and we discuss the most prominent hypotheses aiming to explain the origins and the evolution of autoimmunity in RA.

Different classes of anti-modified protein antibodies are induced on exposure to antigens expressing only one type of modification

ObjectivesAutoantibodies against post-translationally modified proteins (anti-modified protein antibodies or AMPAs) are a hallmark of rheumatoid arthritis (RA). A variety of classes of AMPAs against different modifications on proteins, such as...

Baseline autoantibody profile in rheumatoid arthritis\xa0is associated with early treatment response but not long-term outcomes

BackgroundThe autoantibody profile of seropositive rheumatoid arthritis (RA) is very diverse and consists of various isotypes and antibodies to multiple post-translational modifications. It is yet unknown whether this varying breadth of the autoantibody profile is associated with treatment outcomes. Therefore, we investigated whether the composition of the autoantibody profile in RA, as a marker of the underlying immunopathology, influences initial and long-term treatment outcomes.MethodsIn serum from 399 seropositive patients with RA in the IMPROVED study, drawn at baseline and at the moment of drug tapering, we measured IgG, IgM, and IgA isotypes for anti-cyclic citrullinated peptide-2 and anti‐carbamylated protein antibodies, IgM and IgA rheumatoid factor, and reactivity against four citrullinated and two acetylated peptides (anti-modified protein antibodies (AMPAs)). We investigated the effect of the breadth of the autoantibody profile on (1) change in disease activity score (DAS)44 between 0 and 4 months, (2) initial drug-free remission (DFR, drug-free DAS44 < 1.6) achieved between 1 and 2 years of follow up, and (3) long-term sustained DFR until last follow up.ResultsPatients with a broad autoantibody profile at baseline had a significantly better early treatment response: ΔDAS 0–4 months of 1–2, 3–4, and 5–6 vs 7–8 isotypes, -1.5 (p < 0.001), -1.7 (p = 0.03), and -1.8 (p = 0.04) vs -2.2. Similar results were observed for AMPA number. However, patients with a broad baseline autoantibody profile achieved less initial DFR. For long-term sustained DFR there was no longer an association with the breadth of the autoantibody response. When assessing autoantibodies at the moment of tapering, similar trends were observed.ConclusionsA broad baseline autoantibody profile is associated with a better early treatment response. The breadth of the baseline autoantibody profile, reflecting a break in tolerance against several different autoantigens and extensive isotype switching, may indicate a more active humoral autoimmunity, which could make the underlying disease processes initially more suppressible by medication. The lack of association with long-term sustained DFR suggests that the relevance of the baseline autoantibody profile diminishes over time.Trial registrationISRCTN11916566. Registered on 7 November 2006. EudraCT, 2006- 06186-16. Registered on 16 July 2007.

Breach of autoreactive B cell tolerance by post-translationally modified proteins

ObjectivesOver 50% of patients with rheumatoid arthritis (RA) harbour a variety of anti-modified protein antibodies (AMPA) against different post-translationally modified (PTM) proteins, including anti-carbamylated protein (anti-CarP) antibodies. At present, it...

The contribution of autoantibodies to post-translationally modified proteins to inflammatory arthritis.

This article provides an overview of the recent findings present in the field of antimodified protein antibodies and their relevance for rheumatic disease, with a focus on rheumatoid arthritis. Next to anticitrullinated protein antibodies, also other antimodified protein antibodies such as anticarbamylated protein antibodies or antiacetylated protein antibodies are emerging. Likewise, their possible pathological contributions are getting detailed attention. Their possible relation to disease, both from a clinical as well as biological perspective will be summarized. Current evidence is pointing to the notion that especially a combination of antimodified protein antibodies associates best with clinical phenotype and outcome.

Evolution of autoantibody responses in individuals at risk of rheumatoid arthritis.

Autoantibodies such as rheumatoid factors (RFs), anti-citrullinated protein antibodies (ACPAs), and other anti-modified protein antibodies are important risk factors for the development of rheumatoid arthritis (RA) and probably play an important role in its pathogenesis. In the phase before clinical arthritis becomes apparent, different autoantibody responses can evolve because of increases in their level, isotype switching, affinity maturation, epitope spreading, and a changing glycosylation profile. This evolution may be crucial for the pathogenic properties of the autoantibody responses, and interfering with this process in individuals at risk may become a route to prevent RA. Recent data suggest that interactions between RFs and ACPAs further amplify their inflammatory potential.