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Background:The COVID-19 pandemic led to a rapid increase in remote consultations in rheumatology care. Due to the potential impact of this change on rheumatology clinical training, we investigated trainees’ experiences with telemedicine.Objectives:To assess the impact of telemedicine use during the COVID-19 pandemic on rheumatology training, including supervision.Methods:A voluntary, anonymous web-based survey was administered in English, Spanish, or French from 19/08/2020 to 05/10/2020. Adult and paediatric rheumatology trainees worldwide in training in 2020 were invited to participate via social media and email. Using multiple choice questions, Likert scales, and free text answers, we collected data regarding prior and current telemedicine use, training, and supervision.Results:302 respondents from 33 countries completed the survey, with most (83%, 252/302) in adult rheumatology training. Reported use of telemedicine increased from 13% (39/302) pre-pandemic to 82% (247/302) (Table 1). European trainees predominantly utilised audio-only compared to trainees from the rest of the world (ROW) who predominantly utilised audio-video telemedicine.Most trainees continued to evaluate new patients using telemedicine (65%, 161/247). A larger proportion of trainees were comfortable using telemedicine to evaluate follow-up (69% 170/247) versus new patients (25%, 41/161) (Figure 1).Only 32% (97/302) were trained in telemedicine, with the highest proportion among United States (US) trainees (59%, 69/116); subjects included software, clinical skills, and billing. The majority of trainees found this helpful (92%, 89/97).Supervision was most frequently in the form of verbal discussion after the consultation (Table 1); 24% (59/247) had no telemedicine supervision during the pandemic. In general, trainees found telemedicine negatively impacted their supervision (51%, 123/242) and clinical teaching quality (70%, 171/244); only 9% reported a positive impact on these areas.Conclusion:Adoption of telemedicine during the COVID-19 pandemic has led to areas of concern for rheumatology trainees including inadequate supervision and clinical teaching. Our results suggest a need for education on evaluation of new patients using telemedicine, increasing telemedicine training, and ensuring adequate supervisory arrangements.Table 1.Telemedicine use, supervision, and training by region. Data is presented as n (%). Rest of the world (ROW) data includes Asia (50), Central and South America (23), Canada (12), Australia (8), and Africa (4).Europen = 89USn = 116ROWn = 97Combinedn = 302Telemedicine usePre-pandemic15 (17)9 (8)15 (15)39 (13)During pandemic64 (72)112 (97)71 (73)247 (82)Telemedicine modalitypre-pandemicAudio-only14 (93)3 (33)8 (53)25 (64)Audio-video1 (7)7 (78)7 (47)15 (38)Telemedicine modality during pandemicAudio-only56 (88)47 (42)51 (72)154 (62)Audio-video7 (11)100 (89)29 (41)136 (55)Supervisionpre-pandemicReal-time observation (part of visit)0 (0)4 (44)3 (20)7 (18)Real-time observation (full visit)0 (0)2 (22)2 (13)4 (10)Verbal discussion after8 (53)3 (33)7 (47)18 (46)Written communication after0 (0)0 (0)1 (7)1 (3)None7 (47)2 (22)5 (33)14 (36)Supervision during pandemicReal-time observation (part of visit)2 (3)54 (48)15 (21)71 (29)Real-time observation (full visit)3 (5)32 (29)8 (11)43 (17)Verbal discussion after32 (50)65 (58)28 (39)125 (51)Written communication after7 (11)15 (13)9 (13)31 (13)None28 (44)9 (8)22 (31)59 (24)Figure 1.Rheumatology trainee comfort levels in using telemedicine during the pandemic.Disclosure of Interests:Su-Ann Yeoh: None declared, Kristen Young: None declared, Michael Putman: None declared, Elizabeth Graef: None declared, Francis Berenbaum: None declared, Richard Conway: None declared, Rebecca Grainger Speakers bureau: Speaker fees from Abbvie, Janssen, Novartis, Pfizer, Cornerstones, all not related to this work, Consultant of: Consultancy fees from Abbvie, Janssen, Novartis, Pfizer, Cornerstones, all not related to this work, Grant/research support from: Travel assistance from Pfizer, not related to this work, Adam Kilian: None declared, Maximilian Konig: None declared, Jean Liew Grant/research support from: Research grant from Pfizer unrelated to this manuscript, Pedro M Machado Speakers bureau: Speaker fees from Abbvie, BMS, Celgene, Eli Lilly, Janssen, MSD, Novartis, Orphazyme, Pfizer, Roche and UCB, all unrelated to this manuscript, Consultant of: Consulting fees from Abbvie, BMS, Celgene, Eli Lilly, Janssen, MSD, Novartis, Orphazyme, Pfizer, Roche and UCB, all unrelated to this manuscript, Sebastian E. Sattui: None declared, Jeffrey Sparks Consultant of: Consultancy for Bristol-Myers Squibb, Gilead, Inova Diagnostics, Optum, and Pfizer unrelated to this manuscript, Grant/research support from: Research support from Bristol-Myers Squibb unrelated to this manuscript, Paul Sufka: None declared, Manuel Ugarte-Gil Grant/research support from: Research grants from Janssen and Pfizer unrelated to this manuscript, Laura Upton: None declared, Zachary Wallace: None declared, Jinoos Yazdany Consultant of: Consultancy for Astra Zeneca, Eli Lilly, and Pfizer, not related to this work, Grant/research support from: Research grants from Gilead and Pfizer, not related to this work, Arundathi Jayatilleke: None declared

BackgroundAnti-citrullinated protein antibodies (ACPAs) are the hallmark of rheumatoid arthritis (RA). Protein citrullination is believed to drive autoantigen selection in RA. Nonetheless, several autoantigens in RA are targeted as native (unmodified) proteins. Here, the study of hnRNP A2/B1 (RA33) provides a framework to understand the humoral response to native and citrullinated autoantigens in RA.MethodsRA synovial fluid (SF) cells were analysed by immunoblotting and mass spectrometry. RA33 was cloned from RASF cells and splice variants expressed as recombinant proteins. Antibodies against native and citrullinated RA33 were characterised by ELISA, immunoblotting and immunoprecipitation.ResultsRA33 is citrullinated in the rheumatoid joint and targeted either as a citrullinated or native protein in distinct patient subsets with RA. A novel splice variant (hnRNP B1b) previously associated with disease initiation in experimental arthritis was identified in the RA joint and acts as the major target of the anti-RA33 response. Antibodies exclusively targeting citrullinated RA33 were positively associated with disease duration and erosive disease. In contrast, anti-(native) RA33 antibodies were detected almost exclusively in early RA and identified patients with low radiographic erosion scores. Finally, a unique subset of double-reactive patients demonstrated intermediate severity, but rapid disease progression, suggesting a transitional disease phase in the evolution of an anti-native protein antibody to ACPA response in RA.ConclusionsThese data suggest that native and citrullinated proteins targeted by autoantibodies in RA may be part of a single antibody system and challenge the paradigm of citrullination as the unifying principle underlying loss of tolerance in RA.

We thank Carbillon et al for their correspondence.1 The use of hydroxychloroquine (HCQ) in pregnant women with systemic lupus erythematosus (SLE) is not controversial.2 3 Similar to its primary role in the prophylaxis and treatment of SLE, discontinuation of HCQ in pregnancy has been linked to increased disease activity and glucocorticoid use in women with lupus.4–6 Given its benefit and preferable safety profile, the continuation of baseline HCQ therapy in pregnant women with lupus is recommended to maintain disease remission,2 3 regardless of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) status. In contrast, there is currently no evidence to suggest that baseline use of HCQ in pregnant women with lupus is protective of SARS-CoV-2 infection or severe COVID-19. The authors adequately summarise our findings …

Microbial contributions to the immunopathogenesis of autoimmune rheumatic diseases have been studied since the advent of germ theory in the 19(th) century. With the exception of Group A Streptococcus in rheumatic fever, early studies failed to establish causal relationships between specific pathobionts and rheumatic disease. Today, systemic autoimmune diseases are thought to result from a complex interplay of environmental factors, individual genetic risk, and stochastic events. Interactions of microbiota and the immune system have been shown to promote and sustain chronic inflammation and autoimmunity. In mechanistic studies, microbe-immune cell interactions have been implicated in the initiation of autoimmune rheumatic diseases, e.g. through the posttranslational modification of autoantigens in rheumatoid arthritis or through neutrophil cell death and cross-reactivity with commensal orthologs in systemic lupus erythematosus. In parallel, modern molecular techniques have catalyzed the study of the microbiome in systemic autoimmune diseases. Here, I review current insights gained into the skin, oral, gut, lung, and vascular microbiome in connective tissue diseases and vasculitis. Mechanism relevant to the development and propagation of autoimmunity will be discussed whenever explored. While studies in connective tissue diseases and vasculitis have almost invariably shown abnormal microbiome structure (dysbiosis), substantial variability in microbial composition between studies makes generalization difficult. Moreover, an etiopathogenic role of specific pathobionts cannot be inferred by association alone. Integrating descriptive studies of microbial communities with hypothesis-driven research informed by immunopathogenesis will be important in elucidating targetable mechanisms in preclinical and established rheumatic disease.

Background: Aggregatibacter actinomycetemcomitans (Aa) is a Gram-negative coccobacillus recognized as a pathogen in periodontitis and infective endocarditis. By producing a toxin (leukotoxin A, LtxA) that triggers global hypercitrullination in neutrophils, Aa has been recently linked to rheumatoid arthritis (RA) pathogenesis. Although mechanistic and clinical association studies implicate Aa infection in the initiation of autoimmunity in RA, direct evidence in humans is lacking. Case: We describe a 59-year-old man with anti-citrullinated protein antibody (ACPA)-positive RA who presented for evaluation of refractory disease. He was found to have Aa endocarditis. Following antibiotic treatment, joint symptoms resolved and ACPAs normalized. Given the implications for RA immunopathogenesis, we further investigated the bacterial, genetic and immune factors that may have contributed to the patient's clinical and autoimmune phenotypes. Methods: DNA was extracted from serum and used to amplify the Aa leukotoxin (tx) promoter region by PCR, which was further analyzed by Sanger sequencing. High-resolution identification of HLA alleles was performed by sequenced based typing (SBT). TNF-alpha, IFN-gamma, GM-CSF, IL-1 beta, IL-6, IL-8, IL-17A, IL-18, IL-21, and IL-22 were quantified in serum by a multiplex immunoassay. IgG and IgA antibodies to Aa LtxA were assayed by ELISA. Results: Aa genotyping confirmed infection with a highly leukotoxic strain carrying a 530-bp /tx promoter deletion, shown to result in 10- to 20-fold higher bacterial expression of LtxA. lmmuno-phenotyping showed high anti LtxA antibodies, elevated cytokines implicated in RA pathogenesis (Th1/Th17), and specific host susceptibility conferred by three HLA alleles strongly linked to ACPAs and RA (DRB1*04:04:04, DRB1*15:01, and DPB1*04:01). One year after eradication of Aa, the patient remained free of arthritis and anti-CCP antibodies. Conclusion: In the context of genetic risk for RA, systemic subacute infection with a leukotoxic strain of Aa can drive ACPA production and a clinical phenotype similar to RA.

Objective: Peptidylarginine deiminases (PAD) 2 and 4 are key enzymes in rheumatoid arthritis (RA) pathogenesis due to their ability to generate the protein targets of anti-citrullinated protein antibodies (ACPA). Anti-PAD4 antibodies that cross-react with PAD3 (anti-PAD3/4) have been identified and are associated with severe joint and lung disease. Here, we examined whether anti-PAD2 antibodies were present in patients with RA and defined their clinical significance. Patients and Methods: A PAD2 ELISA was established to screen for anti-PAD2 IgG in sera from RA patients from a prospective observational cohort study (n = 184) and healthy controls (n = 100). RA patient characteristics were compared according to anti-PAD2 antibody status. Multivariable models were constructed to explore the independent associations of anti-PAD2 antibodies with clinical variables. Results: Anti-PAD2 antibodies were found in 18.5% of RA patients and 3% of healthy controls (p < 0.001). Among RA patients, anti-PAD2 antibodies were not associated with traditional genetic or serologic RA risk factors, including HLA-DRβ1 shared epitope alleles, ACPA, rheumatoid factor (RF), or anti-PAD3/4 antibodies. In addition, antibodies to PAD2 were associated with fewer swollen joints, a lower prevalence of interstitial lung disease, and less progression of joint damage. In subset analyses in which patients were stratified by the baseline presence of ACPA/RF or anti-PAD3/4 antibodies, anti-PAD2 antibodies provided additional value in identifying patients with the least progressive joint disease. Conclusions: Anti-PAD2 antibodies represent a novel serologic marker in RA that identifies a genetically and clinically unique subset of patients with less severe joint and lung disease.

Antibodies to leukotoxin A are markers that link Aggregatibacter actinomycetemcomitans –associated periodontitis and rheumatoid arthritis.

ObjectivesThe citrullinating enzyme peptidylarginine deiminase type 4 (PAD4) is the target of a polyclonal group of autoantibodies in patients with rheumatoid arthritis (RA). A subgroup of such antibodies, initially identified by cross-reactivity with peptidylarginine deiminase type 3 (PAD3), is strongly associated with progression of radiographic joint damage and interstitial lung disease and has the unique ability to activate PAD4. The features of these antibodies in terms of their T cell-dependent origin, genetic characteristics and effect of individual antibody specificities on PAD4 function remain to be defined.MethodsWe used PAD4 tagged with the monomeric fluorescent protein mWasabi to isolate PAD4-specific memory B cells from anti-PAD4 positive patients with RA and applied single cell cloning technologies to obtain monoclonal antibodies.ResultsAmong 44 single B cells, we cloned five antibodies with PAD4-activating properties. Sequence analysis, germline reversion experiments and antigen specificity assays suggested that autoantibodies to PAD4 are not polyreactive and arise from PAD4-reactive precursors. Somatic mutations increase the agonistic activity of these antibodies at low calcium concentrations by facilitating their interaction with structural epitopes that modulate calcium-binding site 5 in PAD4.ConclusionsPAD4-activating antibodies directly amplify a key process in disease pathogenesis, making them unique among other autoantibodies in RA. Understanding the molecular basis for their functionality may inform the design of future PAD4 inhibitors.

A bacterial etiology of rheumatoid arthritis (RA) has been suspected since the beginnings of modern germ theory. Recent studies implicate mucosal surfaces as sites of disease initiation. The common occurrence of periodontal dysbiosis in RA suggests that oral pathogens may trigger the production of disease-specific autoantibodies and arthritis in susceptible individuals. We used mass spectrometry to define the microbial composition and antigenic repertoire of gingival crevicular fluid in patients with periodontal disease and healthy controls. Periodontitis was characterized by the presence of citrullinated autoantigens that are primary immune targets in RA. The citrullinome in periodontitis mirrored patterns of hypercitrullination observed in the rheumatoid joint, implicating this mucosal site in RA pathogenesis. Proteomic signatures of several microbial species were detected in hypercitrullinated periodontitis samples. Among these, Aggregatibacter actinomycetemcomitans (Aa), but not other candidate pathogens, induced hypercitrullination in host neutrophils. We identified the pore-forming toxin leukotoxin A (LtxA) as the molecular mechanism by which Aa triggers dysregulated activation of citrullinating enzymes in neutrophils, mimicking membranolytic pathways that sustain autoantigen citrullination in the RA joint. Moreover, LtxA induced changes in neutrophil morphology mimicking extracellular trap formation, thereby releasing the hypercitrullinated cargo. Exposure to leukotoxic Aa strains was confirmed in patients with RA and was associated with both anticitrullinated protein antibodies and rheumatoid factor. The effect of human lymphocyte antigen-DRB1 shared epitope alleles on autoantibody positivity was limited to RA patients who were exposed to Aa These studies identify the periodontal pathogen Aa as a candidate bacterial trigger of autoimmunity in RA.

NETosis, an antimicrobial form of neutrophil cell death, is considered a primary source of citrullinated autoantigens in rheumatoid arthritis (RA) and immunogenic DNA in systemic lupus erythematosus (SLE). Activation of the citrullinating enzyme peptidylarginine deiminase type 4 (PAD4) is believed to be essential for neutrophil extracellular trap (NET) formation and NETosis. PAD4 is therefore viewed as a promising therapeutic target to inhibit the formation of NETs in both diseases. In this review, we examine the evidence for PAD4 activation during NETosis and provide experimental data to suggest that protein citrullination is not a universal feature of NETs. We delineate two distinct biological processes, leukotoxic hypercitrullination (LTH) and defective mitophagy, which have been erroneously classified as “NETosis.” While these NETosis mimics share morphological similarities with NETosis (i.e., extracellular DNA release), they are biologically distinct. As such, these processes can be readily classified by their stimuli, activation of distinct biochemical pathways, the presence of hypercitrullination, and antimicrobial effector function. NETosis is an antimicrobial form of cell death that is NADPH oxidase-dependent and not associated with hypercitrullination. In contrast, LTH is NADPH oxidase-independent and not bactericidal. Rather, LTH represents a bacterial strategy to achieve immune evasion. It is triggered by pore-forming pathways and equivalent signals that cumulate in calcium-dependent hyperactivation of PADs, protein hypercitrullination, and neutrophil death. The generation of citrullinated autoantigens in RA is likely driven by LTH, but not NETosis. Mitochondrial DNA (mtDNA) expulsion, the result of a constitutive defect in mitophagy, represents a second NETosis mimic. In the presence of interferon-α and immune complexes, this process can generate highly interferogenic oxidized mtDNA, which has previously been mistaken for NETosis in SLE. Distinguishing NETosis from LTH and defective mitophagy is paramount to understanding the role of neutrophil damage in immunity and the pathogenesis of human diseases. This provides a framework to design specific inhibitors of these distinct biological processes in human disease.

Dermatomyositis (DM) is characterized by proximal muscle weakness and characteristic skin rash. Pain is a less common feature and usually indicates inflammation of extramuscular structures such as fascia. Here we report a rare case of subscapular bursitis in a 48-year-old woman with DM. She initially presented with severe, sharp, stabbing pain in her right shoulder that worsened with shoulder movement. Magnetic resonance imaging (MRI) revealed inflammation in the right subscapular bursae. A few months later, the patient developed periungual erythema, Gottron's papules, and shawl sign with muscle pain in her thighs. DM was diagnosed based on the presence of interface dermatitis on skin biopsy and diffuse muscle inflammation on MRI. Bursitis and myalgia responded incompletely to nonsteroidal anti-inflammatory drugs but promptly to corticosteroids. Here we report a case of subscapular bursitis as a rare manifestation of DM. Pain in patients with DM may warrant physicians to evaluate for the presence of additional inflammatory processes in the perimuscular structures.

Background Antibodies to citrullinated proteins are a hallmark of rheumatoid arthritis (RA). Porphyromonas gingivalis peptidylarginine deiminase (PPAD) has been implicated in the initiation of RA by generating citrullinated neoantigens and due to its ability to autocitrullinate. Objectives To define the citrullination status and biology of PPAD in P gingivalis and to characterise the anti-PPAD antibody response in RA and associated periodontal disease (PD). Methods PPAD in P gingivalis cells and culture supernatant were analysed by immunoblotting and mass spectrometry to detect citrullination. Recombinant PPAD (rPPAD), inactive mutant PPAD (rPPAD C351S ), and N-terminal truncated PPAD (rPPAD Ntx ) were cloned and expressed in Escherichia coli . Patients with RA and healthy controls were assayed for IgG antibodies to citrullinated rPPAD and unmodified rPPAD C351S by ELISA. Anti-PPAD antibodies were correlated with anti-cyclic citrullinated peptide (third-generation) antibody levels, RA disease activity and PD status. Results PPAD from P gingivalis is truncated at the N-terminal and C-terminal domains and not citrullinated. Only when artificially expressed in E coli , full-length rPPAD, but not truncated (fully active) rPPAD Ntx , is autocitrullinated. Anti-PPAD antibodies show no heightened reactivity to citrullinated rPPAD, but are exclusively directed against the unmodified enzyme. Antibodies against PPAD do not correlate with anti-cyclic citrullinated peptide levels and disease activity in RA. By contrast, anti-PPAD antibody levels are significantly decreased in RA patients with PD. Conclusions PPAD autocitrullination is not the underlying mechanism linking PD and RA. N-terminal processing protects PPAD from autocitrullination and enhances enzyme activity. Anti-PPAD antibodies may have a protective role for the development of PD in patients with RA.

Interstitial lung disease (ILD) is a major cause of death in patients with dermatomyositis (DM). This study was aimed to examine the utility of the erythrocyte sedimentation rate (ESR) as a predictor of ILD and prognostic marker of mortality in patients with DM. One hundred-and-fourteen patients with DM were examined, including 28 with clinically amyopathic DM (CADM). A diagnosis of ILD was made based on high resolution computed tomography (HRCT) scans. The association between elevated ESR and pulmonary impairment and mortality was then examined. ILD was diagnosed in 53 (46.5%) of 114 DM patients. Cancer was diagnosed in 2 (3.8%) of 53 DM patients with ILD and in 24 (92.3%) of those without ILD (P < 0.001). The median ESR (50.0 mm/hour) in patients with ILD was significantly higher than that in patients without ILD (29.0 mm/hour; P < 0.001). ESR was inversely correlated with forced vital capacity (Spearman ρ = - 0.303; P = 0.007) and carbon monoxide diffusing capacity (ρ = - 0.319; P = 0.006). DM patients with baseline ESR ≥ 30 mm/hour had significantly higher mortality than those with ESR < 30 mm/hour (P = 0.002, log-rank test). Patients with a persistently high ESR despite immunosuppressive therapy was associated with higher mortality than those with a normalized ESR (P = 0.039, log-rank test). Elevated ESR is associated with increased mortality in patients with DM due to respiratory failure. Thus, monitoring ESR should be an integral part of the clinical care of DM patients., Graphical Abstract

We thank Dr Quirke and colleagues for their interest in our study. We have some concerns, however, that the authors’ attempt to reconcile our work on Porphyromonas gingivalis peptidylarginine deiminase (PPAD) autocitrullination with their model of periodontal disease in rheumatoid arthritis (RA) misrepresents our data.1 ,2 The discovery that citrullination is the primary antigenic determinant recognised by autoantibodies in RA has generated considerable interest in the family of enzymes, peptidylarginine deiminases (PADs), that catalyse this post-translational protein modification.3 The identification of a bacterial PAD from P gingivalis led to the initial hypothesis by Rosenstein et al 4 that infection with this periodontal pathogen may be aetiologically linked to RA. Two distinct mechanisms have since been hypothesised by which PPAD may trigger loss of tolerance to citrullinated proteins and autoimmunity in RA—first, by acting as a citrullinating enzyme on bacterial and host proteins,4 ,5 and second, through the process of enzyme autocitrullination.6 In the absence of experimental evidence to support PPAD autocitrullination in P gingivalis or in vivo, Quirke et al 2 posited a third mechanism in which PPAD acts as a substrate for human PADs, thereby becoming the target of a citrulline-specific antibody response. The first two hypotheses support P gingivalis as a unique periodontal bacterium relevant to RA pathogenesis. The uniqueness of P gingivalis for RA is lost, however, when considering the third hypothesis of bacterial proteins as putative substrates for human PADs. Under this premise (not supported by experimental data), human PADs may generate citrullinated antigens from any conceivable pathogen, making P gingivalis expendable as a model organism. To strengthen the credibility of PPAD for inducing autoimmunity in RA, Quirke et al variably use these three hypotheses.2 Importantly, our study only addressed the question of PPAD autocitrullination as a possible link …

Background Perforin- and complement-induced cell lysis has recently been implicated in the generation of citrullinated autoantigens in rheumatoid arthritis (RA). By activating PADs in target cells, immune-mediated pore-forming pathways induce abnormal protein citrullination in the RA joint. Here, we demonstrate a novel approach to identify unique autoantibody reactivities to citrullinated proteins generated by the perforin pathway. Objectives To identify novel perforin-induced autoantigens in RA, and to characterize the antibody response to citrullinated hnRNP A1 (cit-hnRNP A1) in patients with RA. Methods 293T cells transiently transfected to express human PAD2 or PAD4 were incubated with perforin from cytotoxic granule contents (GC) to induce PAD activation. Cellular citrullination was confirmed by anti-modified citrulline (AMC) immunoblotting. Inducible autoantigens generated during perforin-mediated cytolysis were identified by immunoblotting using RA patient sera. Antigen identity was determined by 2D gel electrophoresis/mass spectrometry (MS). Recombinant hnRNP A1 was expressed in E. coli. Patients with RA (n=196) from a longitudinal cohort study (ESCAPE RA) and healthy controls (n=56) were assayed for IgG antibodies to unmodified and in vitro citrullinated hnRNP A1 by ELISA. Positivity was defined as an arbitrary unit (AU) above the 98th percentile of controls. Anti-cit-hnRNP A1 antibody specificity was determined by subtracting reactivities measured against unmodified hnRNP A1 from reactivities against cit-hnRNP A1. Results 293T cells expressing PAD2 or PAD4 were used to study PAD-isotype specific autoantigen citrullination. A 34-kDa autoantigen inducible in both PAD2- and PAD4-transfected cells was distinctly targeted by RA patient sera (Fig. 1A; GC: perforin-treated cells, NT: untreated controls). MS identified the modified protein as cit-hnRNP A1, and citrullination by both rPAD2 and rPAD4 was confirmed using purified hnRNP A1. Antibody reactivity against cit-hnRNP A1 was detected in 70 (36%) of RA patients vs. only 1 (2%) of controls (p Conclusions These studies identify hnRNP A1 as a citrullinated autoantigen in RA generated during perforin-mediated cytotoxicity. Anti-cit-hnRNP A1 antibodies may serve as a novel biomarker associated with mechanisms of joint damage in a subset of RA. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3035