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Background Rheumatoid arthritis (RA) is classified as seropositive or seronegative, depending on the presence/absence of rheumatoid factor (RF), primarily IgM RF, and/or anti-citrullinated protein antibodies (ACPA), commonly detected using anti-cyclic citrullinated peptide (CCP) assays. Known risk factors associate with the more severe seropositive form of RA; less is known about seronegative RA. Here, we examine risk factors and clinical phenotypes in relation to presence of autoantibodies in the RA subset that is traditionally defined as seronegative. Methods Anti-CCP2 IgG, 19 ACPA fine-specificities, IgM/IgG/IgA RF, anti-carbamylated-protein (CarP) antibodies, and 17 other autoantibodies, were analysed in 2755 RA patients and 370 controls. Antibody prevalence, levels, and co-occurrence were examined, and associations with risk factors and disease activity during 5 years were investigated for different antibody-defined RA subsets. Results Autoantibodies were detected in a substantial proportion of the traditionally defined seronegative RA subset, with ACPA fine-specificities found in 30%, IgA/IgG RF in 9.4%, and anti-CarP antibodies in 16%, with a 9.6% co-occurrence of at least two types of RA-associated autoantibodies. HLA-DRB1 shared epitope (SE) associated with the presence of ACPA in anti-CCP2-negative RA; in anti-CCP2-positive RA, the SE association was defined by six ACPA fine-specificities with high co-occurrence. Smoking associated with RF, but not with ACPA, in anti-CCP2-negative RA. Presence of ACPA and RF, but not anti-CarP antibodies, in conventionally defined “seronegative” RA, associated with worse clinical outcome. Conclusions “Seronegative” RA is not truly a seronegative disease subset. Additional screening for ACPA fine-specificities and IgA/IgG RF defines a group of patients that resembles seropositive patients with respect to risk factors and clinical picture and may contribute to earlier diagnosis for a subset of anti-CCP2−/IgM RF− patients with a high need for active treatment.

Objectives Porphyromonas gingivalis (P.g.) is discussed to be involved in triggering self-reactive immune responses. The aim of this study was to investigate the autocitrullinated prokaryotic peptidylarginine deiminase (PPAD) from P.g. CH2007 (RACH2007-PPAD) from a rheumatoid arthritis (RA) patient and a synthetic citrullinated PPAD peptide (CPP) containing the main autocitrullination site as potential targets for antibody reactivity in RA and to analyse the possibility of citrullinating native human proteins by PPAD in the context of RA.MethodsRecombinant RACH2007-PPAD was cloned and expressed in Escherichia coli. Purified RACH2007-PPAD and its enzymatic activity was analysed using two-dimensional electrophoresis, mass spectrometry, immunoblot and ELISA. Autoantibody response to different modified proteins and peptides was recorded and bioinformatically evaluated.ResultsRACH2007-PPAD was capable to citrullinate major RA autoantigens, such as fibrinogen, vimentin, hnRNP-A2/B1, histone H1 and multiple peptides, which identify a common RG/RGG consensus motif. 33% of RA patients (n=30) revealed increased reactivity for α-cit-RACH2007-PPAD before RA onset. 77% of RA patients (n=99) presented α-cit-specific signals to CPP amino acids 57–71 which were positively correlated to α-CCP2 antibody levels. Interestingly, 48% of the α-CPP-positives were rheumatoidfactor IgM/anti-citrullinated peptide/protein antibodies (ACPA)-negative. Anti-CPP and α-RACH2007-PPAD antibody levels increase with age. Protein macroarrays that were citrullinated by RACH2007-PPAD and screened with RA patient sera (n=6) and controls (n=4) uncovered 16 RACH2007-PPAD citrullinated RA autoantigens and 9 autoantigens associated with lung diseases. We showed that the α-CPP response could be an important determinant in parenchymal changes in the lung at the time of RA diagnosis (n=106; p=0.018).ConclusionsRACH2007-PPAD induced internal citrullination of major RA autoantigens. Anti-RACH2007-PPAD correlates with ACPA levels and interstitial lung disease autoantigen reactivity, supporting an infection-based concept for induction of ACPAs via enzymatic mimicry.

Background Juvenile idiopathic arthritis is a heterogeneous group of chronic inflammatory joint diseases in children. The presence and role of antibodies targeting citrullinated proteins remains unclear in this patient group. This study aimed to assess antibodies against citrullinated peptides in a Swedish cohort of patients with juvenile idiopathic arthritis and to explore their associations with clinical features and genetic markers. Methods Plasma samples from 334 patients with juvenile idiopathic arthritis were analyzed for antibodies against cyclic citrullinated peptides using enzyme-linked immunosorbent assay. A subset of 100 patients underwent detailed profiling of antibody reactivity to 15 citrullinated peptides and their non-citrullinated counterparts using a multiplex microarray. Rheumatoid factor, antinuclear antibodies, and HLA-DRB1 genotypes were also assessed. Results Antibodies against cyclic citrullinated peptides were detected in 6.2% of patients, with the highest prevalence in the rheumatoid factor–positive polyarthritis subtype. These patients were older at disease onset and more frequently carried HLA-DRB1 shared epitope alleles. Among the 100 profiled patients, 85% of those positive for cyclic citrullinated peptide antibodies showed reactivity to at least one citrullinated peptide, compared to 19% of antibody-negative patients. The most frequent reactivities were directed against peptides derived from filaggrin, fibrinogen, vimentin, and enolase. Reactivity to non-citrullinated peptides was minimal. A significant association was found between reactivity to citrullinated vimentin and the presence of HLA-DRB1 shared epitope alleles. Conclusions This study reveals a distinct pattern of antibody reactivity to citrullinated peptides in a subset of patients with juvenile idiopathic arthritis, suggesting a biologically distinct subgroup with similarities to adult rheumatoid arthritis. These findings identify ACPA-positive JIA as a unique subgroup of young patients and underscore an opportunity to investigate early molecular events driving the immune response to citrullinated proteins and the pathogenesis of arthritis.

Rheumatoid arthritis (RA) associated anti-citrullinated protein autoantibodies (ACPA) target a wide range of modified proteins. Citrullination occurs during physiological processes such as apoptosis, yet little is known about the interaction of ACPA with nuclear antigens or apoptotic cells. Since uncleared apoptotic cells and neutrophil extracellular trap (NET) products have been postulated to be central sources of autoantigen and immunostimulation in autoimmune disease, we sought to characterize the anti-nuclear and anti-neutrophil reactivities of ACPA. Serology showed that a subset of anti-CCP2 seropositive RA patients had high reactivity to full-length citrullinated histones. In contrast, seronegative RA patients displayed elevated IgG reactivity to native histone compared to controls, but no citrulline-specific reactivity. Screening of 10 single B-cell derived monoclonal ACPA from RA patients revealed that four ACPA exhibited strong binding to apoptotic cells and three of these had anti-nuclear (ANA) autoantibody reactivity. Modified histones were confirmed to be the primary targets of this anti-nuclear ACPA subset following immunoprecipitation from apoptotic cell lysates. Monoclonal ACPA were also screened for reactivities against stimulated murine and human neutrophils, and all the nuclear-reactive monoclonal ACPA bound to NETs. Intriguingly, one ACPA mAb displayed a contrasting cytoplasmic perinuclear neutrophil binding and may represent a different NET-reactive ACPA subset. Notably, studies of CRISPR-Cas9 PAD4 KO cells and cells from PAD KO mice showed that the cytoplasmic NET-binding was fully dependent on PAD4, whilst nuclear- and histone-mediated NET reactivity was largely PAD-independent. Our further analysis revealed that the nuclear binding could be explained by consensus-motif driven ACPA cross-reactivity to acetylated histones. Specific acetylated histone peptides targeted by the monoclonal antibodies were identified and the anti-modified protein autoantibody (AMPA) profile of the ACPA was found to correlate with the functional activity of the antibodies. In conclusion, when investigating monoclonal ACPA, we could group ACPA into distinct subsets based on their nuclear binding-patterns and acetylation-mediated binding to apoptotic cells, neutrophils, and NETs. Differential anti-modified protein reactivities of RA-autoantibody subsets could have an important functional impact and provide insights in RA pathogenesis.

Background There is a need for biomarker to identify patients “at risk” for rheumatoid arthritis (risk-RA) and to better predict the therapeutic response and in this study we tested the hypothesis that novel native and citrullinated heterogeneous nuclear ribonucleoprotein (hnRNP)-DL autoantibodies could be possible biomarkers. Methods Using protein macroarray and ELISA, epitope recognition against hnRNP-DL was analysed in sera from different developed RA disease and diagnosed SLE patients. Toll-like receptor (TLR) 7/9 and myeloid differentiation primary response gene 88 (MyD88)-dependency were studied in sera from murine disease models. HnRNP-DL expression in cultivated cells and synovial tissue was analysed by indirect immunofluorescence, immunoblot and immunohistochemistry. Results HnRNP-DL was highly expressed in stress granules, citrullinated in the rheumatoid joint and targeted by autoantibodies either as native or citrullinated proteins in patient subsets with different developed RA disease. Structural citrullination dependent epitopes (SCEs) of hnRNP-DL were detected in 58% of the SLE patients although 98% of these sera were α-CCP-2-negative. To obtain a specific citrullinated signal value, we subtracted the native antibody value from the citrullinated signal. The citrullinated/native index of autoantibodies against hnRNP-DL (CN DL -Index) was identified as a new value for an “individual window of treatment success” in early RA and for the detection of RF IgM/α-CCP-2 seronegative RA patients (24–46%). Negative CN DL -index was found in SLE patients, risk-RA and early RA cohorts such as EIRA where the majority of these patients are DAS28-responders to methotrexate (MTX) treatment (87%). High positive CN DL -values were associated with more severe RA, shared epitope and parenchymal changes in the lung. Specifically, native α-hnRNP-DL is TLR7/9-dependent, associated with pain and ROC analysis revealed an association to initial MTX or etanercept treatment response, especially in seronegative RA patients. Conclusion CN DL -index defines people at risk to develop RA and the “window of treatment success” thereby closing the sensitivity gap in RA.

We constructed miniaturized autoantigen arrays to perform large-scale multiplex characterization of autoantibody responses directed against structurally diverse autoantigens, using submicroliter quantities of clinical samples. Autoantigen microarrays were produced by attaching hundreds of proteins, peptides and other biomolecules to the surface of derivatized glass slides using a robotic arrayer. Arrays were incubated with patient serum, and spectrally resolvable fluorescent labels were used to detect autoantibody binding to specific autoantigens on the array. We describe and characterize arrays containing the major autoantigens in eight distinct human autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis. This represents the first report of application of such technology to multiple human disease sera, and will enable validated detection of antibodies recognizing autoantigens including proteins, peptides, enzyme complexes, ribonucleoprotein complexes, DNA and post-translationally modified antigens. Autoantigen microarrays represent a powerful tool to study the specificity and pathogenesis of autoantibody responses, and to identify and define relevant autoantigens in human autoimmune diseases.

The immune system protects us from foreign substances or pathogens by generating specific antibodies. The variety of immunoglobulin (Ig) paratopes for antigen recognition is a result of the V(D)J rearrangement mechanism, while a fast and efficient immune response is mediated by specific immunoglobulin isotypes obtained through class switch recombination (CSR). To get a better understanding on how antibody-based immune protection works and how it changes with age, the interdependency between these two parameters need to be addressed. Here, we have performed an in depth analysis of antibody repertoires of 14 healthy donors representing different gender and age groups. For this task, we developed a unique pyrosequencing approach, which is able to monitor the expression levels of all immunoglobulin V(D)J recombinations of all isotypes including subtypes in an unbiased and quantitative manner. Our results show that donors have individual immunoglobulin repertoires and cannot be clustered according to V(D)J recombination patterns, neither by age nor gender. However, after incorporating isotype-specific analysis and considering CSR information into hierarchical clustering the situation changes. For the first time the donors cluster according to age and separate into young adults and elderly donors (>50). As a direct consequence, this clustering defines the onset of immune senescence at the age of fifty and beyond. The observed age-dependent reduction of CSR ability proposes a feasible explanation why reduced efficacy of vaccination is seen in the elderly and implies that novel vaccine strategies for the elderly should include the \"Golden Agers\".

ObjectivesTo investigate how individual rheumatoid arthritis (RA) autoantibodies associate with individual signs and symptoms at the time of RA diagnosis.MethodsIgA, IgG, IgM rheumatoid factor (RF), antibodies against cyclic citrullinated peptide version 2 (anti-CCP2) and 16 individual antibodies against citrullinated protein (ACPA) reactivities were analysed centrally in baseline sera from 1600 patients with RA classified according to the 1987 American College of Rheumatology (ACR) criteria. These results were related to C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), number of swollen and tender joints (SJC and TJC), 28-joint disease activity scores (DAS28 and DAS28CRP), global disease activity evaluated by the patients and Health Assessment Questionnaire, all obtained at baseline.ResultsIndividually, all autoantibodies except immunoglobulin G (IgG) RF associated with low SJC and TJC and with high ESR. In IgM RF-negative patients, ACPA associated strictly with low number of swollen and tender joints. This association persisted in multiple regression and stratified analyses where IgM and IgA RF instead associated with inflammation expressed as ESR. Among subjects without any ACPA peptide reactivity, there was no association between RF isotypes and ESR. The effect of RF on ESR increased with the number of ACPA reactivities, especially for IgM RF. In patients fulfilling the 1987 ACR criteria without taking RF into account, associations between IgM RF and high ESR, as well as between ACPA and low joint counts, remained.ConclusionWhereas ACPA associate with low counts of affected joints in early RA, RF associates with elevated measures of systemic inflammation in an ACPA-dependent manner. This latter finding corroborates in vitro models of ACPA and RF in immune complex-induced inflammation. These phenotypic associations are independent of classification criteria.

IntroductionThe second generation anticycliccitrullinated peptide (anti-CCP2) assay detects the majority but not all anticitrullinated protein/peptide antibodies (ACPA). Anti-CCP2-positive rheumatoid arthritis (RA) is associated with HLA-DRB1* shared epitope (SE) alleles and smoking. Using a multiplex assay to detect multiple specific ACPA, we have investigated the fine specificity of individual ACPA responses and the biological impact of additional ACPA reactivity among anti-CCP2-negative patients.MethodsWe investigated 2825 patients with RA and 551 healthy controls with full data on anti-CCP2, HLA-DRB1* alleles and smoking history concerning reactivity against 16 citrullinated peptides and arginine control peptides with a multiplex array.ResultsThe prevalence of the 16 ACPA specificities ranged from 9% to 58%. When reactivity to arginine peptides was subtracted, the mean diagnostic sensitivity increased by 3.2% with maintained 98% specificity. Of the anti-CCP2-negative patients, 16% were found to be ACPA positive. All ACPA specificities associated with SE, and all but one with smoking. Correction for arginine reactivity also conveyed a stronger association with SE for 13/16 peptides. Importantly, when all ACPA specificities were analysed together, SE and smoking associated with RA in synergy among ACPA positive, but not among ACPA-negative subjects also in the anti-CCP2-negative subset.ConclusionsMultiplexing detects an enlarged group of ACPA-positive but anti-CCP2-negative patients with genetic and environmental attributes previously assigned to anti-CCP2-positive patients. The individual correction for arginine peptide reactivity confers both higher diagnostic sensitivity and stronger association to SE than gross ACPA measurement.