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Additional file 2: Supplementary Figure 2. Comparisons of pentameric C-reactive protein (pCRP) (A–C), monomeric (m)CRP (D–F), and mCRP/pCRP ratio (G–I) demonstrated between normal vs. subnormal estimated glomerular filtration rate (eGFR) (A, D, G), presence vs. absence of hematuria (B, E, H) and presence vs. absence albuminuria (C, F, I) in patients with ANCA-associated vasculitis. Abnormal eGFR

Additional file 3: Supplementary Figure 3. Standard curves and reactivity of the monoclonal detection antibody 8C10 towards monomeric C-reactive protein (mCRP) and pentameric (p)CRP: (A) standard curve for 8C10 antibody with mCRP; (B) reactivity of 8C10 antibody with pCRP; and (C) standard curve for samples in the present study.

Additional file 1: Supplementary Figure 1. Comparisons of pentameric C-reactive protein (pCRP) (A–D), monomeric (m)CRP (E–H), and mCRP/pCRP ratio (I–L) in 160 patients with systemic lupus erythematosus with lupus nephritis vs. no nephritis (A, E, I), normal vs. subnormal estimated glomerular filtration rate (eGFR) (B, F, J), presence vs. absence of hematuria (C, G, K) and presence vs. absence of albuminuria (D, H, L). Abnormal eGFR

Additional file 3: Supplementary Figure 3. Standard curves and reactivity of the monoclonal detection antibody 8C10 towards monomeric C-reactive protein (mCRP) and pentameric (p)CRP: (A) standard curve for 8C10 antibody with mCRP; (B) reactivity of 8C10 antibody with pCRP; and (C) standard curve for samples in the present study.

BackgroundThere 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.MethodsUsing 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. ResultshnRNP-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-CCP2-negative. To obtain a specific citrullinated signal value, we subtracted the native antibody value from the citrullinated signal. This CNDL (Citrullinated-Native-hnRNP-DL)-index identified and the bioinformatic value was explored, as a new value for an “individual window of treatment success” in early RA and for the detection of RF-IgM/α-CCP2 seronegative RA patients (24-46%). Negative CNDL-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 CNDL-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.ConclusionCNDL-index defines patients is a possible biomarker for develop RA and the “window of treatment success” thereby potentially closing the sensitivity gap.

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 (CNDL-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 CNDL-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 CNDL-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 CNDL-index defines people at risk to develop RA and the “window of treatment success” thereby closing the sensitivity gap in RA. Correction in: Arthritis Research & Therapy, Volume 23, Issue 1, Article Number 255, DOI 10.1186/s13075-021-02639-z

Patients with chronic inflammatory diseases are often treated with immunosuppressants and therefore are of particular concern during the SARS-CoV-2 pandemic. Serological tests will improve our understanding of the infection and immunity in this population, unless they tests give false positive results. The aim of this study was to evaluate the specificity of SARS-Cov-2 serological assays using samples from patients with chronic inflammatory diseases collected prior to April 2019, thus defined as negative. Samples from patients with multiple sclerosis (MS, n=10), rheumatoid arthritis (RA, n=47) with or without rheumatoid factor (RF) and/or anti-cyclic citrullinated peptide antibodies (anti-CCP2) and systemic lupus erythematosus (SLE, n=10) with or without RF, were analyzed for SARS-CoV-2 antibodies using 17 commercially available lateral flow assays (LFA), two ELISA kits and one in-house developed IgG multiplex bead-based assay. Six LFA and the in-house validated IgG assay correctly produced negative results for all samples. However, the majority of assays (n=13), gave false positive signal for samples from patients with RA and SLE. This was most notable in samples from RF positive RA patients. No false positive samples were detected in any assay using samples from patients with MS. Poor specificity of commercial serological assays could possibly be, at least partly, due to interfering antibodies in samples from patients with chronic inflammatory diseases. For these patients, the risk of false positivity should be considered when interpreting results of the SARS-CoV-2 serological assays.

Supplemental material, sj-pdf-1-lup-10.1177_09612033211014570 for Associations with thrombosis are stronger for antiphosphatidylserine/prothrombin antibodies than for the Sydney criteria antiphospholipid antibody tests in SLE by Sahwa Elbagir, Giorgia Grosso, NasrEldeen A Mohammed, Amir I Elshafie, Elnour M Elagib, Agneta Zickert, Vivek Anand Manivel, Eleftheria Pertsinidou, Musa A. M Nur, Iva Gunnarsson, Johan Rönnelid and Elisabet Svenungsson in Lupus

Supplemental material, sj-pdf-2-lup-10.1177_09612033211014570 for Associations with thrombosis are stronger for antiphosphatidylserine/prothrombin antibodies than for the Sydney criteria antiphospholipid antibody tests in SLE by Sahwa Elbagir, Giorgia Grosso, NasrEldeen A Mohammed, Amir I Elshafie, Elnour M Elagib, Agneta Zickert, Vivek Anand Manivel, Eleftheria Pertsinidou, Musa A. M Nur, Iva Gunnarsson, Johan Rönnelid and Elisabet Svenungsson in Lupus

Seropositive rheumatoid arthritis (RA) is characterized by the presence of rheumatoid factor (RF) and anti-citrullinated protein autoantibodies (ACPA) with different fine-specificities. Yet, other serum anti-modified protein autoantibodies (AMPA), e.g. anti-carbamylated (Carb), -acetylated (KAc), and malondialdehyde acetaldehyde (MAA) modified protein antibodies, have been described. In this comprehensive study, we analyze 30 different IgG and IgA AMPA reactivities to Cit, Carb, KAc, and MAA antigens detected by ELISA and autoantigen arrays in N=1985 newly diagnosed RA patients. Association with patient characteristics such as smoking and disease activity were explored. Carb and KAc reactivities by different assays were primarily seen in patients also positive for anti-citrulline reactivity. Modified vimentin (mod-Vim) peptides were used for direct comparison of different AMPA reactivities, revealing that IgA AMPA recognizing mod-Vim was mainly detected in subsets of patients with high IgG anti-Cit-Vim levels and a history of smoking. IgG reactivity to acetylation was mainly detected in a subset of patients with Cit and Carb reactivity. Anti-acetylated histone reactivity was RA-specific and associated with high anti-CCP2 IgG levels, multiple ACPA fine-specificities, and smoking status. This reactivity was also found to be present in CCP2+ RA-risk individuals without arthritis. Our data further demonstrate that IgG autoreactivity to MAA was increased in RA compared to controls with highest levels in CCP2+ RA, but was not RA-specific, and showed low correlation with other AMPA. Anti-MAA was instead associated with disease activity and was not significantly increased in CCP2+ individuals at risk of RA. Notably, RA patients could be subdivided into four different subsets based on their AMPA IgG and IgA reactivity profiles. Our serology results were complemented by screening of monoclonal antibodies derived from single B cells from RA patients for the same antigens as the RA cohort. Certain CCP2+ clones had Carb or Carb+KAc+ multireactivity, while such reactivities were not found in CCP2- clones. We conclude that autoantibodies exhibiting different patterns of ACPA fine-specificities as well as Carb and KAc reactivity are present in RA and may be derived from multireactive B-cell clones. Carb and KAc could be considered reactivities within the "Cit-umbrella" similar to ACPA fine-specificities, while MAA reactivity is distinctly different.

We investigated whether belimumab treatment impacts on levels of autoantibodies and cytokines of interest in systemic lupus erythematosus (SLE). Longitudinally collected serum samples from 78 belimumab-treated Swedish SLE patients were analysed. Serum cytokine levels were determined using Luminex xMAP technology, and nuclear antigen autoantibody specificities using addressable laser bead immunoassay. In patients with detectable levels at baseline, interferon (IFN)-a2 levels were lower at month 6 (median; interquartile range (IQR): 8.9; 1.5-54.9 pg/mL) versus baseline (28.4; 20.9-100.3 pg/mL; p = 0.043). Interleukin (IL)-6 (baseline: 7.1; 2.9-16.1 pg/mL) decreased from month 6 (0.5; 0.5-6.3 pg/mL; p = 0.018) and throughout a 24 month follow-up. IL-10 (baseline: 12.6; 2.8-29.7 pg/mL) showed more rapid decreases from month 3 (1.8; 0.6-9.1 pg/mL; p = 0.003). Levels of anti-dsDNA (p < 0.001), anti-Smith antigen (Sm) (p = 0.002), anti-U1 small nuclear ribonucleoprotein (U1RNP) (p < 0.001), anti-Sm-U1RNP complex (p = 0.028), and anti-ribosomal P (p = 0.012) antibodies decreased from month 3 and remained decreased. Anti-Sm positivity at baseline was associated with higher probability and/or shorter time to achieve sustained SLE responder index-4 response (hazard ratio (HR): 2.52; 95% CI: 1.20-5.29; p = 0.015), independently of other factors. Decline of IL-6 levels through month 3 was greater in responders. In summary, belimumab treatment lowered IFN-a2, IL-6, and IL-10 levels, as well as levels of multiple autoantibodies, however after different time spans. Notably, anti-Sm positivity and early decline in IL-6 levels were associated with favorable treatment outcome. Funding agencies: The Swedish Research Council; Swedish RheumatismAssociation (R-859621; R-862161; R-844801); Professor Nanna Svartz Foundation (2018-00250); Ulla and Roland Gustafsson Foundation (2019-12); King Gustaf V’s 80-year Foundation; Swedish Society of Medicine; Ingegerd Johansson Donation; King Gustaf V and Queen Victoria’s Foundation of Freemasons; Region Östergötland (ALF grants); Region Stockholm; Region Uppsala; Region Skåne; Faculty of Medicine, Lund University; AlfredÖsterlund’s Foundation; Anna-Greta Crafoord Foundation; Greta and Johan Kock’s Foundation; Skåne UniversityHospital; and the Karolinska Institutet Foundations.

Supplemental material, sj-pdf-1-lup-10.1177_0961203320945387 for High IgA antiphospholipid autoantibodies in healthy Sudanese explain the increased prevalence among Sudanese compared to Swedish systemic lupus erythematosus patients by Sahwa Elbagir, Amir I Elshafie, Elnour M Elagib, NasrEldeen A Mohammed, Mawahib IE Aledrissy, Vivek Anand Manivel, Eleftheria Pertsinidou, Musa AM Nur, Iva Gunnarsson, Elisabet Svenungsson and Johan Rönnelid in Lupus

Additional file 3: Supplementary Table 3. Baseline characteristics in CCP2-positive and -negative RA. The table show baseline characteristics for the EIRA patients included in the study, based on anti-CCP2 IgG status; p-values indicate differences between subsets with respect to: age, female-to-male ratio, number of smokers, HLA-DRB1 SE-positivity, PTPN22-positivity, DAS28 and CRP.

Additional file 7: Supplementary Table 6. Associations between different ACPA fine-specificities and HLA-DRB1 SE, in anti-CCP2-positive and anti-CCP2-negative RA. Odds ratios with 95% confidence intervals are shown for associations between HLA-DRB1 SE and presence/absence of 19 different ACPA fine-specificities, in anti-CCP2-positive and -negative RA: p-values indicate differences in ORs between ACPA fine-specificity-positive and -negative subsets in the anti-CCP2-positive subset.

Additional file 6: Supplementary Table 5. Associations between different autoantibodies and PTPN22 polymorphism, in anti-CCP2-negative RA. Odds ratios with 95% confidence intervals are shown for associations between PTPN22 polymorphism and presence/absence of ACPA, RF, other autoantibodies, or "any autoantibody" in anti-CCP2-negative RA.

Additional file 6: Supplementary Table 5. Associations between different autoantibodies and PTPN22 polymorphism, in anti-CCP2-negative RA. Odds ratios with 95% confidence intervals are shown for associations between PTPN22 polymorphism and presence/absence of ACPA, RF, other autoantibodies, or "any autoantibody" in anti-CCP2-negative RA.

Additional file 7: Supplementary Table 6. Associations between different ACPA fine-specificities and HLA-DRB1 SE, in anti-CCP2-positive and anti-CCP2-negative RA. Odds ratios with 95% confidence intervals are shown for associations between HLA-DRB1 SE and presence/absence of 19 different ACPA fine-specificities, in anti-CCP2-positive and -negative RA: p-values indicate differences in ORs between ACPA fine-specificity-positive and -negative subsets in the anti-CCP2-positive subset.

Additional file 4: Supplementary Figure 1. ACPA fine-specificity levels in anti-CCP2-negative RA and controls. ACPA levels for 19 individual ACPA fine-specificities are shown in separate graphs with AU values on the Y-axes. Only ACPA levels above cutoff for positivity are shown.

Additional file 1.

Additional file 4: Supplementary Figure 1. ACPA fine-specificity levels in anti-CCP2-negative RA and controls. ACPA levels for 19 individual ACPA fine-specificities are shown in separate graphs with AU values on the Y-axes. Only ACPA levels above cutoff for positivity are shown.

Additional file 8: Supplementary Table 7. Associations between RF isotypes and smoking, in anti-CCP2-positive and anti-CCP2-negative RA. Odds ratios with 95% confidence intervals are shown for associations between smoking and presence/absence of IgM, IgG or IgA RF, in anti-CCP2-positive and -negative RA; p-values indicate differences in ORs between RF isotype-positive and -negative subsets.

Additional file 8: Supplementary Table 7. Associations between RF isotypes and smoking, in anti-CCP2-positive and anti-CCP2-negative RA. Odds ratios with 95% confidence intervals are shown for associations between smoking and presence/absence of IgM, IgG or IgA RF, in anti-CCP2-positive and -negative RA; p-values indicate differences in ORs between RF isotype-positive and -negative subsets.

International audience; Introduction : Individual patients with rheumatoid arthritis (RA) show divergent specific anti-citrullinated protein/peptide antibodies (ACPA) patterns, but hitherto no individual ACPA specificity has consistently been linked to RA pathogenesis. ACPA are also implicated in immune complexes (IC)-associated joint pathology, but until now, there has been no method to investigate the role of individual ACPA in RA IC formation and IC-associated pathogenesis.Methods : We have developed a new technique based on IC binding to C1q-coated magnetic beads to purify and solubilise circulating IC in sera and synovial fluids (SF) from 77 patients with RA. This was combined with measurement of 19 individual ACPA in serum, SF and in the IC fractions from serum and SF. We investigated whether occurrence of individual ACPA as well as number of ACPA in these compartments was related to clinical and laboratory measures of disease activity and inflammation.Results : The majority of individual ACPA reactivities were enriched in SF as compared with in serum, and levels of ACPA in IC were regulated independently of levels in serum and SF. No individual ACPA reactivity in any compartment showed a dominating association to clinical and laboratory measures of disease activity and severity. Instead, the number of individual ACPA reactivities in the IC fraction from SF associated with a number of markers of joint destruction and inflammation. Conclusions : Our data highlight the polyclonality of ACPA in joint IC and the possibility that a broad ACPA repertoire in synovial fluid IC might drive the local inflammatory and matrix-degrading processes in joints, in analogy with antibody-induced rodent arthritis models.

Objective Antifibrillar collagen type II (anti-CII) antibody-positive patients with rheumatoid arthritis (RA) have early but not late signs of increased inflammation and joint erosions. We wanted to replicate this in a large RA cohort, and to relate to human leukocyte antigen (HLA)-DRB1* alleles. Methods Anti-CII and anti-cyclic citrullinated peptide (CCP)2 were measured at baseline in 773 patients with RA from the Swedish Epidemiological Investigation in Rheumatoid Arthritis (EIRA) study with clinical follow-up data from the Swedish Rheumatology Quality Register (SRQ) registry, and 1476 with HLA-DRB1* information. Comparisons were done concerning C reactive protein (CRP), erythrocyte sedimentation rate (ESR), tender joint count (TJC), swollen joint count (SJC), Disease Activity Score encompassing 28 joints based on ESR (DAS28), DAS28CRP, pain-Visual Analogue Scale (VAS), global-VAS and Health Assessment Questionnaire Score (HAQ) at eight occasions during 5 years, and association with HLA-DRB1* alleles. Results Anti-CII associated with elevated CRP, ESR, SJC, DAS28 and DAS28CRP at diagnosis and up to 6 months, whereas anti-CCP2 associated with SJC and DAS28 from 6 months to 5 years, but not earlier. The anti-CII-associated phenotype was strong, and predominated in anti-CII/anti-CCP2 double-positive patients. Anti-CII was associated with improvements in CRP, ESR, SJC, TJC and DAS28, whereas anti-CCP2 was associated with deteriorations in SJC and DAS28 over time. Anti-CII-positive patients achieved European League Against Rheumatism good or moderate response more often than negative patients. Anti-CII was positively associated with HLA-DRB1*01 and HLA-DRB1*03, with significant interaction, and double-positive individuals had >14 times higher mean anti-CII levels than HLA double negatives. Whereas smoking was associated with elevated anti-CCP2 levels, smokers had lower anti-CII levels. Conclusions Anti-CII seropositive RA represents a distinct phenotype, in many respects representing the converse to the clinical, genetic and smoking associations described for anticitrullinated protein peptide autoantibodies. Although not diagnostically useful, early anti-CII determinations predict favourable inflammatory outcome in RA.

Illustration of a conditional inference tree for developing RA in pre-symptomatic individuals compared with control subjects. CCP2 anti-CCP2 antibodies, Filaggrin anti-filaggrin antibodies, CEP-1 = anti-α-enolase antibodies, IgM-RF immunoglobulin M rheumatoid factor, HLA-SE human leukocyte antigen shared epitope, n number of individuals, Pr probability of detecting a pre-symptomatic individual, + = positive, − = negative. (DOCX 29 kb)

Prevalence, sensitivity, specificity and OR of numbers of RF isotypes, alone and/or in combination with ten different ACPA specificities, anti-CCP2 antibodies and anti-carbamylated protein antibodies in pre-symptomatic individuals and population controls. (DOCX 17kb)

Illustration of a conditional inference tree for developing RA in pre-symptomatic individuals compared with control subjects. CCP2 anti-CCP2 antibodies, Filaggrin anti-filaggrin antibodies, CEP-1 = anti-α-enolase antibodies, IgM-RF immunoglobulin M rheumatoid factor, HLA-SE human leukocyte antigen shared epitope, n number of individuals, Pr probability of detecting a pre-symptomatic individual, + = positive, − = negative. (DOCX 29 kb)

Circulating antinuclear antibodies (ANA) are commonly present in the systemic autoimmune disease Systemic Lupus Erythematosus (SLE) and in other systemic rheumatic diseases, in humans as well as in dogs. The indirect immunofluorescence (IIF)-ANA test is the standard method for detecting ANA. Further testing for specific ANA with immunoblot techniques or ELISAs is routinely performed in humans to aid in the diagnosis and monitoring of disease. Several specific ANA identified in humans have been identified also in suspected canine SLE but, in contrast to humans, investigation of autoantibodies in canine SLE is mainly restricted to the IIF-ANA test. Our aim was to identify both known and novel specific ANA in dogs and to investigate if different IIF-ANA patterns are associated with different specific ANA in dogs. Sera from 240 dogs with suspicion of autoimmune disease (210 IIF-ANA positive (ANA(pos)) and 30 IIF-ANA negative (ANA(neg))) as well as sera from 27 healthy controls were included. The samples were analysed with a line immunoassay, LIA (Euroline ANA Profile 5, Euroimmun, Lubeck, Germany) and four different ELISAs (Euroimmun). The ANA(pos) dogs were divided in two groups depending on the type of IIF-ANA pattern. Of the 210 ANA(pos) samples 68 were classified as ANA homogenous (ANA(H)) and 141 as ANA speckled (ANA(S)), one sample was not possible to classify. Dogs in the ANA(H) group had, compared to the other groups, most frequently high levels of anti-double stranded deoxyribonucleic acid (dsDNA) and anti-nucleosome ANA. Anti-dsDNA antibodies were confirmed in some dogs with the Crithidia luciliae indirect immunofluorescence test (CLIFT).The frequency of ANA(H) dogs with values above those observed in the healthy group was significantly higher compared to ANA(S) dogs for anti-dsDNA, anti-nucleosome, and anti-histone reactivity. Dogs in the ANA(S) group had, compared to the other groups, most frequently high levels of anti-ribonucleoproteins (RNP) and/or anti-Smith (Sm) antibodies. Reactivity against Sjogren's syndrome related antigens (SS)-A (including the Ro-60 and Ro-52 subcomponents), SS-B, histidyl tRNA synthetase (Jo-1), topoisomerase I antigen (Scl-70), polymyositis-scleroderma antigen (PM-Scl) and proliferating cell nuclear antigen (PCNA) was also noted in individual dogs. In conclusion, by using a commercial LIA and different ELISAs originally developed for detection of human ANA, we identified several specific ANA in serum samples from dogs sampled for IIF-ANA testing. Further, we found that the types of IIF-ANA pattern were associated with reactivity against some particular nuclear antigens. (C) 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

IFN-γ was measured in supernatants after in vitro stimulation of peripheral blood mononuclear cells with collagen type II (CII), purified protein derivative or influenza virus. IFN-γ production in response to CII was similar in rheumatoid arthritis (RA) patients and healthy control individuals. The IFN-γ response to purified protein derivative and influenza virus was lower in RA patients, reflecting a general T-cell hyporesponsiveness in RA. After recalculating the response to CII taking this hyporesponsiveness into account the CII response was higher in RA patients, and was associated with human leucocyte antigen (HLA)-DRB1*0401 and HLA-DQA1*0301-DQB1*0302 (HLA-DQ8). Rheumatoid arthritis patients with elevated serum levels of immunoglobulin (Ig)G anti-CII antibodies had lower CII-induced IFN-γ production than patients with low anti-CII levels. The relative increase in CII-reactivity in RA patients as compared with healthy control individuals, and the association of a higher response with RA-associated HLA haplotypes, suggest the existence of a potentially pathogenic cellular reactivity against CII in RA., Introduction: Despite much work over past decades, whether antigen-specific immune reactions occur in rheumatoid arthritis (RA) and to what extent such reactions are directed towards joint-specific autoantigens is still questionable. One strong indicator for antigenic involvement in RA is the fact that certain major histocompatibility complex (MHC) class II genotypes [human leucocyte antigen (HLA)-DR4 and HLA-DR1] predispose for the development of the disease [1]. In the present report, collagen type II (CII) was studied as a putative autoantigen on the basis of both clinical and experimental data that show an increased frequency of antibodies to CII in RA patients [2,3,4] and that show that CII can induce experimental arthritis [5]. It is evident from the literature that RA peripheral blood mononuclear cells (PBMCs) respond poorly to antigenic stimulation [6,7,8], and in particular evidence for a partial tolerization to CII has been presented [9]. The strategy of the present work has accordingly been to reinvestigate T-cell reactivity to CII in RA patients, to relate it to the response to commonly used recall antigens and to analyze IFN-γ responses as an alternative to proliferative responses. Aims: To study cellular immune reactivity to CII in patients with RA and in healthy control individuals and to correlate this reactivity to HLA class II genotypes and to the presence of antibodies to CII in serum. Methods: Forty-five patients who met the 1987 American College of Rheumatology classification criteria for RA [10] and 25 healthy control individuals of similar age and sex were included. Twenty-six of these patients who had low levels of anti-CII in serum were randomly chosen, whereas 19 patients with high anti-CII levels were identified by enzyme-linked immunosorbent assay (ELISA)-screening of 400 RA sera. Heparinized blood was density gradient separated and PBMCs were cultured at 1 × 106/ml in RPMI-10% fetal calf serum with or without antigenic stimulation: native or denatured CII (100 μ g/ml), killed influenza virus (Vaxigrip, Pasteur Mérieux, Lyon, France; diluted 1 : 1000) or purified protein derivative (PPD; 10 μ g/ml). CII was heat-denatured in 56°C for 30 min. Cell supernatants were collected after 7days and IFN-γ contents were analyzed using ELISA. HLA-DR and HLA-DQ genotyping was performed utilizing a polymerase chain reaction-based technique with sequence-specific oligonucleotide probe hybridization. Nonparametric statistical analyses were utilized throughout the study. Results: PBMCs from both RA patients and healthy control individuals responded with inteferon-γ production to the same degree to stimulation with native and denatured CII (Fig. 1a), giving median stimulation indexes with native CII of 4.6 for RA patients and 5.4 for healthy control individuals, and with denatured CII of 2.9 for RA patients and 2.6 for healthy control individuals. RA patients with elevated levels of anti-CII had a weaker IFN-γ response to both native and denatured CII than did healthy control individuals (P = 0.02 and 0.04, respectively). Stimulation with the standard recall antigens PPD and killed influenza virus yielded a median stimulation index with PPD of 10.0 for RA patients and 51.3 for healthy control individuals and with influenza of 12.3 for RA patients and 25.7 for healthy, control individuals. The RA patients displayed markedly lower responsiveness to both PPD and killed influenza virus than did healthy control individuals (Fig. 1b). IFN-γ responses to all antigens were abrogated when coincubating with antibodies blocking MHC class II. The low response to PPD and killed influenza virus in RA patients relative to that of healthy control individuals reflects a general downregulation of antigen-induced responsiveness of T cells from RA patients [6,7,8]. That no difference between the RA group and the control group was recorded in CII-induced IFN-γ production therefore indicates that there may be an underlying increased responsiveness to CII in RA patients, which is obscured by the general downregulation of T-cell responsiveness in these patients. In order to address this possibility, we calculated the fraction between individual values for the CII-induced IFN-γ production and the PPD-induced and killed influenza virus-induced IFN-γ production, and compared these fractions. A highly significant difference between the RA and healthy control groups was apparent after stimulation with both native CII and denatured CII when expressing the response as a fraction of that with PPD (Fig. 2a). Similar data were obtained using killed influenza virus-stimulated IFN-γ values as the denominator (Fig. 2b). When comparing the compensated IFN-γ response to denatured CII stimulation between RA patients with different HLA genotypes, highly significant differences were evident, with HLA-DRB1*0401 patients having greater CII responsiveness than patients who lacked this genotype (Fig. 3a). HLA-DQ8 positive patients also displayed a high responsiveness to CII as compared with HLA-DQ8 negative RA patients (Fig. 3b). These associations between the relative T-cell reactivity to denatured CII and HLA class II genotypes were not seen in healthy control individuals. Similar results were achieved using influenza as denominator (P = 0.02 for HLA-DRB1*0401 and P = 0.01 for HLA-DQ8). Discussion: No reports have previously systematically taken the general T-cell hyporesponsiveness in RA into account when investigating specific T-cell responses in this disease. In order to address this issue we used the T-cell responses to PPD and killed influenza virus as reference antigens. This was made on the assumption that exposure to these antigens is similar in age-matched and sex-matched groups of RA patients and healthy control individuals. The concept of a general hyporesponsiveness in RA T cells has been documented in several previous reports, in which both nominal antigens [6,7,8] and mitogens [11,12,13] have been used. The fact that a similar functional downregulation in RA PBMCs was obtained with both PPD and killed influenza virus as reference antigens strengthens the validity of our approach. We identified an association between the IFN-γ response to CII and HLA-DRB1*0401 and HLA-DQ8 in the RA patient group, which is of obvious interest because both these MHC class II alleles have been associated with high responsiveness to CII in transgenic mice that express these human MHC class II molecules [14,15]. There was no association between high anti-CII levels and shared epitope (HLA-DRB1*0401 or HLA-DRB1*0404). Conclusion: CII, a major autoantigen candidate in RA, can elicit an IFN-γ response in vitro that is associated with HLA-DRB1*0401 and HLA-DQ8 in RA patients. This study, with a partly new methodological approach to a classical problem in RA, has provided some additional support to the notion that CII may be a target autoantigen of importance for a substantial group of RA patients. Continued efforts to identify mechanisms behind the general hyporesponsiveness to antigens in RA, as well as the mechanisms behind the potential partial anergy to CII, may provide us with better opportunities to study the specificity and pathophysiological relevance of anti-CII reactivity in RA.

BackgroundRheumatoid arthritis (RA) can be divided into two major subsets based on the presence or absence of antibodies to citrullinated peptide antigens (ACPA). Until now, data from genome-wide association studies (GWAS) have only been published from ACPA-positive subsets of RA or from studies that have not separated the two subsets. The aim of the current study is to provide and compare GWAS data for both subsets.Methods and resultsGWAS using the Illumina 300K chip was performed for 774 ACPA-negative patients with RA, 1147 ACPA-positive patients with RA and 1079 controls from the Swedish population-based case-control study EIRA. Imputation was performed which allowed comparisons using 1,723,056 single nucleotide polymorphisms (SNPs). No SNP achieved genome-wide significance (2.9 × 10⁻⁸) in the comparison between ACPA-negative RA and controls. A case-case association study was then performed between ACPA-negative and ACPA-positive RA groups. The major difference in this analysis was in the HLA region where 768 HLA SNPs passed the threshold for genome-wide significance whereas additional contrasting SNPs did not reach genome-wide significance. However, one SNP close to the RPS12P4 locus in chromosome 2 reached a p value of 2 × 10⁶ and this locus can thus be considered as a tentative candidate locus for ACPA-negative RA.ConclusionsACPA-positive and ACPA-negative RA display significant risk allele frequency differences which are mainly confined to the HLA region. The data provide further support for distinct genetic aetiologies of RA subsets and emphasise the need to consider them separately in genetic as well as functional studies of this disease.

Immune complexes (ICs) can induce production of cytokines by peripheral blood mononuclear cells via Fc receptors. Rheumatoid factor (RF) develop in response to ICs in many clinical and experimental settings. We investigated whether and how polyethylene glycol (PEG) precipitated ICs from rheumatoid arthritis (RA) sera and synovial fluid (SF) can influence cytokine production by peripheral blood mononuclear cells. We also examined the relationship between RF and IC induced cytokine production. Parallel sera and SF from 47 RA patients and sera from 15 healthy control individuals were PEG precipitated. The precipitates were added to serum-free peripheral blood mononuclear cell cultures and tumour necrosis factor (TNF)-alpha levels were measured after 20 hours. In separate cell culture experiments FcgammaRIIa and FcgammaRIII were blocked and monocytes were depleted or enriched. RF in serum was determined by nephelometry, and IgG levels in precipitates and anti-cyclic citrullinated peptide antibodies in serum were measured using ELISA. Clinical data were collected from the patients' charts. In two separate investigations, we demonstrated a correlation between RF, PEG-precipitated IgG levels and induction of the proinflammatory cytokine TNF-alpha by PEG-precipitated SF ICs. No such correlation was found for serum ICs. TNF-alpha levels induced by SF precipitates, but not serum precipitates, correlated with the number of swollen and tender joints. Monocytes/macrophages were shown to be the main responder cells, and blockade of FcgammaRIIa, but not blockade of FcgammaRIII, inhibited TNF-alpha production in cultures stimulated with precipitated ICs. Anti-cyclic citrullinated peptide correlated with RF but exhibited no association with IgG content in PEG precipitates or with precipitate-induced TNF-alpha levels. These findings support the hypothesis that SF ICs and correlated RF production are directly linked to cytokine-dependent inflammation in RA. Suppression of monocytes/macrophages in RA joints or blockade of the primate-specific activating FcgammaRIIa receptor might be ways to reduce IC-induced TNF-alpha production in the joints of seropositive RA patients.

Figure S1. The correlation between RF-IgM and all proteins analyzed. (PDF 284 kb)

Additional file 1: Supplementary Table 1. Citrullinated peptide antigens on the multiplex microarray. The table lists citrullinated antigens used on the multiplex microarray, including name, protein of origin, and amino acid sequence, as well as references.

A more detailed description of the MS proteomic method is described. (PDF 289 kb)

Based on the epidemiological link between periodontitis and rheumatoid arthritis (RA), and the unique feature of the periodontal bacterium Porphyromonas gingivalis to citrullinate proteins, it has been suggested that production of anti-citrullinated protein antibodies (ACPA), which are present in a majority of RA patients, may be triggered in the gum mucosa. To address this hypothesis, we investigated the antibody response to a citrullinated P. gingivalis peptide in relation to the autoimmune ACPA response in early RA, and examined citrulline-reactivity in monoclonal antibodies derived from human gingival B cells. Antibodies to a citrullinated peptide derived from P. gingivalis (denoted CPP3) and human citrullinated peptides were analyzed by multiplex array in 2,807 RA patients and 372 controls; associations with RA risk factors and clinical features were examined. B cells from inflamed gingival tissue were single-cell sorted, and immunoglobulin (Ig) genes were amplified, sequenced, cloned and expressed (n=63) as recombinant monoclonal antibodies, and assayed for citrulline-reactivities by enzyme-linked immunosorbent assay. Additionally, affinity-purified polyclonal anti-cyclic-citrullinated peptide (CCP2) IgG, and monoclonal antibodies derived from RA blood and synovial fluid B cells (n=175), were screened for CPP3-reactivity. Elevated anti-CPP3 antibody levels were detected in RA (11%), mainly CCP2+ RA, compared to controls (2%), p, Frontiers in Immunology, 13, ISSN:1664-3224

A more detailed description of the affinity-based proteomic method is described. (PDF 370 kb)

Additional file 5: Supplementary Table 4. Other autoantibodies in anti-CCP2-positive and -negative RA and controls. Frequencies of 17 other autoantibodies in anti-CCP2-positive RA, anti-CCP2-negative RA and controls are shown, as well as median antibody levels in anti-CCP2-positive and -negative RA (p-values indicate differences between anti-CCP2-negative and anti-CCP2-positive RA subsets).

Table S2. Medications for the patients are reported. (PDF 526 kb)

Additional file 9: Supplementary Figure 2. Antibody levels in EIRA RA cases based on smoking status. IgM, IgG and IgA RF levels, as well as anti-CCP2 IgG levels, are shown for never, current and former smokers in all EIRA RA patients where smoking data was available.

Additional file 1: Supplementary Table 1. Citrullinated peptide antigens on the multiplex microarray. The table lists citrullinated antigens used on the multiplex microarray, including name, protein of origin, and amino acid sequence, as well as references.

Additional file 5: Supplementary Table 4. Other autoantibodies in anti-CCP2-positive and -negative RA and controls. Frequencies of 17 other autoantibodies in anti-CCP2-positive RA, anti-CCP2-negative RA and controls are shown, as well as median antibody levels in anti-CCP2-positive and -negative RA (p-values indicate differences between anti-CCP2-negative and anti-CCP2-positive RA subsets).

Additional file 2: Supplementary Table 2. Other (non-citrullinated) autoantigens on the multiplex microarray. The table lists the other antigens used on the multiplex microarray, including name, protein of origin, and major associated disease(s).

Figure S1. The correlation between RF-IgM and all proteins analyzed. (PDF 284 kb)

Additional file 9: Supplementary Figure 2. Antibody levels in EIRA RA cases based on smoking status. IgM, IgG and IgA RF levels, as well as anti-CCP2 IgG levels, are shown for never, current and former smokers in all EIRA RA patients where smoking data was available.

Additional file 1: Figure 1. Sequence, structure and major immunogenic region (mir) of hnRNP-D and hnRNP-DL. A, Schematic representation of hnRNP-D (isoform p45), hnRNP-DL and the different recombinant hnRNP-DL variants studied. The main structural features are highlighted. Mir-region is the major immunogenic region, RBD1 and RBD2 are RNA-binding domains 1 and 2, Gly-rich is the C-terminal glycine-rich region of the proteins. B, Global amino acid sequence alignment of hnRNP-D and hnRNP-DL1 (isoform 1). HnRNP-D and -DL share 89.1% similarity by sequenc e[1]. Regions “mir”, “RBD1”, “RBD2” and “Gly-rich” are highlighted. Figure 2. Characterisation of autoantibodies against, A, citrullinated α-hnRNP-DLmir (cit-DL), B, α-hnRNP-DLmir (DL) and C, ∆OD between cit-DL and DL (ΔDL) determed by ELISA in sera of other diseases (n=127; MS n=20, reA n=7, Sclero n=20, Sjö n=20, PsA n=20, MB n=20, OA n=20). The dotted lines markes the cutoff vs. other diseases (except systemic lupus erythematosus) or healthy controls with 98% specificity each. OD, optical density; nm, nano meter; vs., versus; MS, multiple sclerosis; reA, reactive arthritis; Sclero, scleroderma; Sjö, Sjögren´s syndrome; PsA, psoriasis arthritis; MB, ankylosing spondylitis; OA. Osteoarthritis. Table 1. Mann Whitney U-test of (cit) α-hnRNP-DLmir-OD signals of seropositive and seronegative data sets of RA-cohorts. Table 2. Mann Whitney U-test of cit α-hnRNP-DLmir-OD signals of seronegative data sets of RA-cohorts and data sets of other inflammatory diseases. Figure 3. XY-Plot and Spearman Correlation of citrullinated or native α-hnRNP-DLmir versus ΔhnRNP-DLmir for the early RA cohort EIRA (A/D; n=404), the seropositive EIRA sera (B/E; n=202) and the seronegative EIRA sera (C/F; n=202). Table 3. Spearman correlation of the early RA sera of the EIRA cohort (n=404). The results are given as R value (left of slash) with the corresponding p-value (right of slash). Table 4. Spearman correlation of the 242 EIRA sera treated with MTX (α-CCP2 positive n=133, α-CCP2 negative n=109). The results are given as R value (left of slash) with the corresponding p-value (right of slash). Table 5. Spearman correlation of the established RA sera of the Predict cohort (n=94; RF IgM and/or α-CCP2 positive n=64, RF IgM and α-CCP2 negative n=30). The results are given as R value (left of slash) with the corresponding p-value (right of slash). Table 6. ROC analysis of native hnRNP-DLmir of MTX-treated EIRA patients (n=192; seropositive n=93, seronegative n=99). Table 7. Negative CNDL-index of MTX-treated EIRA patients n=192 (Resp. n=161, non-Resp. n=31). Table 8. ROC analysis of native hnRNP-DLmir of Enbrel®-treated Predict patients (n=94; seropositive n=63, seronegative n=31). Figure 4. High baseline titer against α-hnRNP-DLmir (DL) is rather present in 6-month EULAR Responder RA patients who had received MTX or α-TNF inhibitor therapy (Enbrel®). A-C, Citrullinated α-hnRNP-DLmir (citDL) (A), α-hnRNP-DLmir (DL) (B) and ∆ OD between citDL and DL (ΔDL) (C) were measured by ELISA in patient sera from the EIRA cohort treated with MTX (n=192) with 161 EULAR Responder and 31 EULAR non-Responder among 6 months. The evaluation was done according to the cutoff versus other diseases. D, α-DL were measured by ELISA in patient sera from the Predict cohort treated with α-TNF inhibitor therapy with 6-month EULAR response data (n=94, responder n=63, non-Responder n=31). Based on the signals, a response-cutoff (dotted line, OD 0.174) was determined, from which only responders are recognized as positive. OD, optical density; nm, nano meter; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; MTX, Methotrexate; Resp., 6-month EULAR Responder. Figure 5. A, Influence of cytokines on hnRNP-DL expression determined by immunoblotting. Cellular extracts from unstimulated, IL1α- or TNFα-stimulated HeLa cells and from unstimulated and IL6-stimulated HepG2 cells were probed with α-hnRNP-DL1/2-peptide specific rabbit serum. B, Citrullination of hnRNP-DL in synovial tissue from a patient with rheumatoid arthritis was investigated with an α-deiminated arginine antibody and an α-hnRNP-DL antibody. Both positive bands were labled with hnRNP-DL, which isoforms were not analysed.

Table S1. The number of missing data points for each variable is shown. (PDF 668 kb)

Additional file 1: Figure 1. Sequence, structure and major immunogenic region (mir) of hnRNP-D and hnRNP-DL. A, Schematic representation of hnRNP-D (isoform p45), hnRNP-DL and the different recombinant hnRNP-DL variants studied. The main structural features are highlighted. Mir-region is the major immunogenic region, RBD1 and RBD2 are RNA-binding domains 1 and 2, Gly-rich is the C-terminal glycine-rich region of the proteins. B, Global amino acid sequence alignment of hnRNP-D and hnRNP-DL1 (isoform 1). HnRNP-D and -DL share 89.1% similarity by sequenc e[1]. Regions “mir”, “RBD1”, “RBD2” and “Gly-rich” are highlighted. Figure 2. Characterisation of autoantibodies against, A, citrullinated α-hnRNP-DLmir (cit-DL), B, α-hnRNP-DLmir (DL) and C, ∆OD between cit-DL and DL (ΔDL) determed by ELISA in sera of other diseases (n=127; MS n=20, reA n=7, Sclero n=20, Sjö n=20, PsA n=20, MB n=20, OA n=20). The dotted lines markes the cutoff vs. other diseases (except systemic lupus erythematosus) or healthy controls with 98% specificity each. OD, optical density; nm, nano meter; vs., versus; MS, multiple sclerosis; reA, reactive arthritis; Sclero, scleroderma; Sjö, Sjögren´s syndrome; PsA, psoriasis arthritis; MB, ankylosing spondylitis; OA. Osteoarthritis. Table 1. Mann Whitney U-test of (cit) α-hnRNP-DLmir-OD signals of seropositive and seronegative data sets of RA-cohorts. Table 2. Mann Whitney U-test of cit α-hnRNP-DLmir-OD signals of seronegative data sets of RA-cohorts and data sets of other inflammatory diseases. Figure 3. XY-Plot and Spearman Correlation of citrullinated or native α-hnRNP-DLmir versus ΔhnRNP-DLmir for the early RA cohort EIRA (A/D; n=404), the seropositive EIRA sera (B/E; n=202) and the seronegative EIRA sera (C/F; n=202). Table 3. Spearman correlation of the early RA sera of the EIRA cohort (n=404). The results are given as R value (left of slash) with the corresponding p-value (right of slash). Table 4. Spearman correlation of the 242 EIRA sera treated with MTX (α-CCP2 positive n=133, α-CCP2 negative n=109). The results are given as R value (left of slash) with the corresponding p-value (right of slash). Table 5. Spearman correlation of the established RA sera of the Predict cohort (n=94; RF IgM and/or α-CCP2 positive n=64, RF IgM and α-CCP2 negative n=30). The results are given as R value (left of slash) with the corresponding p-value (right of slash). Table 6. ROC analysis of native hnRNP-DLmir of MTX-treated EIRA patients (n=192; seropositive n=93, seronegative n=99). Table 7. Negative CNDL-index of MTX-treated EIRA patients n=192 (Resp. n=161, non-Resp. n=31). Table 8. ROC analysis of native hnRNP-DLmir of Enbrel®-treated Predict patients (n=94; seropositive n=63, seronegative n=31). Figure 4. High baseline titer against α-hnRNP-DLmir (DL) is rather present in 6-month EULAR Responder RA patients who had received MTX or α-TNF inhibitor therapy (Enbrel®). A-C, Citrullinated α-hnRNP-DLmir (citDL) (A), α-hnRNP-DLmir (DL) (B) and ∆ OD between citDL and DL (ΔDL) (C) were measured by ELISA in patient sera from the EIRA cohort treated with MTX (n=192) with 161 EULAR Responder and 31 EULAR non-Responder among 6 months. The evaluation was done according to the cutoff versus other diseases. D, α-DL were measured by ELISA in patient sera from the Predict cohort treated with α-TNF inhibitor therapy with 6-month EULAR response data (n=94, responder n=63, non-Responder n=31). Based on the signals, a response-cutoff (dotted line, OD 0.174) was determined, from which only responders are recognized as positive. OD, optical density; nm, nano meter; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; MTX, Methotrexate; Resp., 6-month EULAR Responder. Figure 5. A, Influence of cytokines on hnRNP-DL expression determined by immunoblotting. Cellular extracts from unstimulated, IL1α- or TNFα-stimulated HeLa cells and from unstimulated and IL6-stimulated HepG2 cells were probed with α-hnRNP-DL1/2-peptide specific rabbit serum. B, Citrullination of hnRNP-DL in synovial tissue from a patient with rheumatoid arthritis was investigated with an α-deiminated arginine antibody and an α-hnRNP-DL antibody. Both positive bands were labled with hnRNP-DL, which isoforms were not analysed.

Table S1. The number of missing data points for each variable is shown. (PDF 668 kb)

A more detailed description of the affinity-based proteomic method is described. (PDF 370 kb)