Search

Rheumatoid arthritis (RA) synovitis is dominated by monocytes/macrophages with inflammatory patterns resembling microbial stimulation. In search of triggers, we reduced the intestinal microbiome in 20 RA patients (open label study DRKS00014097) by bowel cleansing and 7-day fasting (≤250 kcal/day) and performed immune monitoring and microbiome sequencing. Patients with metabolic syndrome (n = 10) served as a non-inflammatory control group. Scores of disease activity (DAS28/SDAI) declined within a few days and were improved in 19 of 20 RA patients after breaking the fast (median ∆DAS28 = −1.23; ∆SDAI = −43%) or even achieved remission (DAS28 < 2.6/n = 6; SDAI < 3.3/n = 3). Cytometric profiling with 46 different surface markers revealed the most pronounced phenomenon in RA to be an initially increased monocyte turnover, which improved within a few days after microbiota reduction and fasting. Serum levels of IL-6 and zonulin, an indicator of mucosal barrier disruption, decreased significantly. Endogenous cortisol levels increased during fasting but were insufficient to explain the marked improvement. Sequencing of the intestinal microbiota indicated that fasting reduced potentially arthritogenic bacteria and changed the microbial composition to species with broader metabolic capabilities. More eukaryotic, predominantly fungal colonizers were observed in RA, suggesting possible involvement. This study demonstrates a direct link between the intestinal microbiota and RA-specific inflammation that could be etiologically relevant and would support targeted nutritional interventions against gut dysbiosis as a causal therapeutic approach.

Objectives To assess the association between venous thromboembolic (VTE) events and autoantibodies, following patients from RA diagnosis, measuring occurrence, levels and collective load of different autoantibodies against post-translational protein modifications, in particular recognizing citrullination (e.g. citrullinated fibrinogen) and RF by isotype. Methods A cohort of 2814 patients with newly diagnosed RA were followed for incident VTE through register linkages. Sera from RA diagnosis were centrally analysed for antibodies to second generation cyclic citrullinated peptides (anti-CCP2), 20 anti-citrullinated protein antibody (ACPA) fine-specificities, antibodies to additional protein modifications (carbamylation and acetylation) and RF by isotype. Association between baseline serology status and future VTE was analysed using Cox regression adjusted for age, sex and calendar period of RA diagnosis, overall and stratified by anti-CCP2 and RF positivity. Results During a median 16 years of follow-up, 213 first-ever VTE events were registered (5.0/1000 person-years). IgG anti-CCP2 (present in 65% of cohort) associated with VTE (hazard ratio [HR] = 1.33, 95% CI: 1.00, 1.78), in a dose-response manner. The risk of VTE increased with number of ACPA fine-specificities. IgM RF, but no other RF isotypes, associated with VTE (HR = 1.38, 95% CI: 1.04, 1.82). The associations were independent from smoking and HLA-DRB1 shared epitope alleles. None of the carbamylated or acetylated antibody reactivities associated with VTE. Conclusion Anti-CCP2, load of ACPA fine-specificities and IgM RF at RA diagnosis are associated with an increased risk of future VTE in RA. Antibodies to citrullinated fibrinogen did not differ substantially from other ACPA fine-specificities. Autoreactivity to other post-translational modifications was not associated with VTE risk.

Publisher's version (útgefin grein), Objective: To investigate the relationship between anti–citrullinated protein antibodies (ACPAs), specific ACPA subspecificities, rheumatoid factor (RF) isotypes, and incident cardiovascular (CV) events in patients with rheumatoid arthritis (RA). Methods: Serum samples from Swedish patients with new-onset RA (diagnosed within 1 year of symptom onset between 1996 and 2009) were centrally typed for anti–cyclic citrullinated peptide 2 (anti-CCP2) antibodies, 20 ACPA subspecificities, and RF isotypes. Patients were followed up longitudinally in nationwide registers to monitor the occurrence of acute coronary syndrome (ACS), stroke, CV-related death, and major adverse CV events (MACE). The association between each serologic marker and CV outcome, and the impact of adjustment for the Disease Activity Score in 28 joints (DAS28), smoking status, and income at baseline, were assessed using Cox proportional hazards models. In addition, associations of serologic markers with all-cause mortality were explored. Results: In total, 2,814 patients with RA were included in the study. The median follow-up was 13 years, during which the CV end points of ACS, stroke, or CV-related death were reported to occur in 375 patients. Occurrence and/or levels of anti-CCP2 were associated with risk of incident ACS (hazard ratio [HR] 1.46, 95% confidence interval [95% CI] 1.03–2.06), stroke (HR 1.47, 95% CI 1.03–2.10), CV-related death (P = 0.024 for association with anti-CCP2 levels), and MACE (HR 1.34, 95% CI 1.06–1.70). Similarly, an association with the number of ACPA subspecificities was observed; however, this could not be attributed to any individual or group of ACPA subspecificities. Presence of IgM-RF was associated with all CV end points except ACS, and IgA-RF was exclusively associated with CV-related death. Adjustment for smoking status, income, and DAS28 scores decreased most of the HRs, whereas IgA-RF remained associated with CV-related death (HR 1.61, 95% CI 1.05–2.48). All of the assessed serologic makers were associated with all-cause mortality. Conclusion: RF isotypes and ACPAs are associated with future CV events in patients with RA. ACPA levels and number of subspecificities seem more important than the occurrence of particular subspecificities, and these associations were not explained by a history of ever smoking., Karolinska institutet (strategic research area epidemiology), The nordic research council (nordforsk), The rheumatology research foundation (foreum), Stiftelsen för Strategisk Forskning, Vetenskapsrådet, Stockholms Läns Landsting, The swedish heart lung foundation

The emergence of SARS-CoV-2 led to pandemic spread of coronavirus disease 2019 (COVID-19), manifesting with respiratory symptoms and multi-organ dysfunction. Detailed characterization of virus-neutralizing antibodies and target epitopes is needed to understand COVID-19 pathophysiology and guide immunization strategies. Among 598 human monoclonal antibodies (mAbs) from ten COVID-19 patients, we identified 40 strongly neutralizing mAbs. The most potent mAb CV07-209 neutralized authentic SARS-CoV-2 with IC50 of 3.1 ng/ml. Crystal structures of two mAbs in complex with the SARS-CoV-2 receptor-binding domain at 2.55 and 2.70 Å revealed a direct block of ACE2 attachment. Interestingly, some of the near-germline SARS-CoV-2 neutralizing mAbs reacted with mammalian self-antigens. Prophylactic and therapeutic application of CV07-209 protected hamsters from SARS-CoV-2 infection, weight loss and lung pathology. Our results show that non-self-reactive virus-neutralizing mAbs elicited during SARS-CoV-2 infection are a promising therapeutic strategy., HIGHLIGHTS • Characterization of potent human monoclonal SARS-CoV-2 neutralizing antibodies • Some SARS-CoV-2 antibodies reacted with mammalian self-antigens in different organs • Crystal structures of two antibodies in complex with SARS-CoV-2 RBD at 2.55/2.70 Å • Post-exposure antibody treatment protected from lung damage in infected hamsters, Kreye et al. report the isolation and characterization of monoclonal antibodies isolated from COVID-19 patients, some of which were found to display autoreactivity with mammalian self-antigens in different organs. Crystal structures of two antibodies in complex with SARS-CoV-2 Spike RBD reveal antibody engagement with the ACE2 binding site from different approach angles. One antibody is further evaluated for in vivo efficacy and was found to be both protective and efficacious post-challenge in a hamster infection model.

IntroductionAnticitrullinated peptide antibody (ACPA) responses for 22 citrullinated peptides in patients with early rheumatoid arthritis (RA) were analysed and related to radiological and clinical outcome during the first 2 years in a prospective inception cohort.MethodsThe ACPA reactivities were assessed in 1022 patients with early RA (symptoms ResultsFrequency of ACPA reactivities varied between 13.3% and 63.1%. Of the anticyclic citrullinated peptide-2 (anti-CCP2) antibody-negative patients, ACPA reactivities were positive in 32.6%. Smoking, human leucocyte antigen-shared epitope (HLA-SE), anti-CCP2/rheumatoid factor, protein tyrosine phosphatase non-receptor type 22 (1858C/T) and DAS28 were significantly associated with number of ACPA reactivities. The ACPA reactivities modified differently the development of DAS28 over 24 months (identified using trajectories). Anti-Filaggrin307-324, anti-hnRNP (Peptide)-Z1 and anti-F4-CIT-R antibodies anticipated lower DAS28 values (pConclusionsSeveral ACPA reactivities modified significantly the DAS28 development during the first 24 months and were significantly associated with Larsen score at baseline, 24 months and radiological progression.

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.

Chronic inflammation is a major trigger of local and systemic bone loss. Disintegration of cell–matrix interaction is a prerequisite for the invasion of inflammatory tissue into bone. CD44 is a type I transmembrane glycoprotein that connects a variety of extracellular matrix proteins to the cell surface. Tumor necrosis factor (TNF) is a major inducer of chronic inflammation and its overexpression leads to chronic inflammatory arthritis. By generating CD44−/− human TNF-transgenic (hTNFtg) mice, we show that destruction of joints and progressive crippling is far more severe in hTNFtg mice lacking CD44, which also develop severe generalized osteopenia. Mutant mice exhibit an increased bone resorption due to enhanced number, size, and resorptive capacity of osteoclasts, whereas bone formation and osteoblast differentiation are not affected. Responsiveness of CD44-deficient osteoclasts toward TNF is enhanced and associated with increased activation of the p38 mitogen-activated protein kinase. These data identify CD44 as a critical inhibitor of TNF-driven joint destruction and inflammatory bone loss.

Autoimmune diseases are characterised and regularly diagnosed by the presence of autoantibodies to heterogeneous nuclear RNP (hnRNP) autoantigens. These proteins represent a group of autoantigens which are targeted by autoantibodies from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and mixed connective tissue disease. The authors investigated a new member of the hnRNP family targeted by autoantibodies and tested a combination of hnRNPs in humans and …

International audience; This study was conducted with sera from patients with rheumatoid arthritis, systemic lupus erythematosus as well as with arthritis and lupus-like disease animal models to identify identical autoantigens in human and animal models for disease modifying use.

Autoantibodies directed against spliceosomal heterogeneous nuclear ribonucleoproteins (hnRNPs) are a typical feature of rheumatoid arthritis, systemic lupus erythematosus, and mixed-connective tissue disease. With the aim of investigating a potential pathogenic role of these Abs, we have studied the Ab response to A2/B1 hnRNPs in different murine models of lupus. The specificity of anti-A2/B1 Abs was tested with a series of 14 overlapping synthetic peptides covering the region 1–206 of A2 that contains most of the epitopes recognized by patients’ Abs. A major epitope recognized very early during the course of the disease by Abs from most of MRL lpr/lpr mice but not from other lupus mice and from mice of different MHC haplotypes immunized against B1 was identified in residues 50–70. This peptide contains a highly conserved sequence RGFGFVTF also present in other hnRNPs and small nuclear ribonucleoproteins. Abs reacting with a second A2 epitope identified in residues 35–55 were detectable several weeks later, suggesting an intramolecular B cell epitope spreading during the course of the disease. We identified several T cell epitopes within the region 35–175 that generated an effective Th cell response with IL-2 and IFN-γ secretion in nonautoimmune CBA/J mice sharing the same MHC haplotype H-2k as MRL/lpr mice. None of the peptides stimulated T cells primed in vivo with B1. Because Abs to peptide 50–70 were detected significantly earlier than Abs reacting with other A2 peptides and the protein itself, it is possible that within the protein, this segment contains residues playing an initiator role in the induction of the anti-A2/B1 and antispliceosome Ab response.

RA33 is a nuclear autoantigen with an apparent molecular mass of 33 kD. Autoantibodies against RA33 are found in about 30% of sera from RA patients, but only occasionally in sera from patients with other connective tissue diseases. To characterize RA33, the antigen was purified from HeLa cell nuclear extracts to more than 90% homogeneity by affinity chromatography on heparin-Sepharose and by chromatofocusing. Sequence analysis of five tryptic peptides revealed that their sequences matched corresponding sequences of the A2 protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex. Furthermore, RA33 was shown to be present in the 40S hnRNP complex and to behave indistinguishably from A2 in binding to single stranded DNA. In summary, these data strongly indicate that RA33 and A2 are the same protein, and thus identify on a molecular level a new autoantigen.

The heterogeneus nuclear ribonucleoprotein (hnRNP) D, which is also known as AU-rich element binding factor 1 (AUF1), decreases stability of many short-lived mRNAs (including mRNAs of IL-1 and TNFa) by binding to adenosine and uridine rich sequences (ARE) contained in their 3'-untranslated regions. Previous studies had indicated this protein to be recognized by sera from patients RA, SLE and MCTD. To investigate this autoimmune response in greater detail 356 sera from patients (pts) with various rheumatic disorders were tested by immunoblotting employing the recombinant 45 kDa variant of D/AUF1 (the largest of the 4 known D/AUF1 proteins). Autoantibodies (aab) were detected in 20% of RA pts (n = 105), 34% of SLE pts (n = 70), 17% of MCTD pts (n = 31) and in 25% of pts with primary Sjoegren's syndrome (n = 21), but in less than 5% of 129 pts with other rheumatic disorders and not at all in healthy controls. Importantly, anti-D/AUF1 aab were already present in 25% of 60 patients with early RA of less than 6 months duration, whereas only one of 40 non-RA patients with other forms of early arthritis showed this antibody. Epitope mapping studies showed the aab to be directed to conformational epitopes in the N-terminal part of hnRNP-D/AUF1 known to be indispensable for the protein's function. However, aab did not interfere with RNA binding as assessed by gel shift assays employing the ARE of the TNFa mRNA. Instead, they were able to supershift protein-RNA complexes indicating binding sites for RNA and aab to be distinct. Thus, these findings suggest that anti-D/AUF1 aab target the mRNA decay complex which may form another large ribonucleoprotein target structure in systemic autoimmunity. We are tempted to speculate that increased formation of such complexes (e.g. due to overexpression of instable mRNAs such as those for IL-1 and TNFa as seen in RA) may lead to pathologic autoimmune reactions against D/AUF1 and other proteins of mRNA decay complexes.

In an attempt elucidate the role of autoimmune processes in the pathogenesis of rheumatoid arthritis (RA) we investigated the T cell responses to two autoantigens targeted by autoantibodies of patients with RA, (i) the heterogeneous nuclear ribonucleoprotein (hnRNP) A2/RA33 and (ii) filaggrin which is one of the target structures recognized by anti-citrulline antibodies. Stimulation assays were performed with peripheral blood mononuclear cells of 50 RA patients, 20 patients with osteoarthritis and 21 healthy control individuals using recombinant hnRNP-A2/RA33 as well as some fragments thereof and recombinant filaggrin both in unmodified and citrullinated form. Antigen-specific T cell clones (TCC) were obtained by cultivating T cell lines in the presence of antigen and IL-2 followed by limiting-dilution cloning. Proliferative responses to hnRNP-A2/RA33 were seen in 60% of the RA patients with a mean stimulation index (SI) of 3.5 ± 2.8 and were significantly higher than those observed in the control group (mean SI=1.7 ± 1, P < 0.00005). There was no correlation with the presence of anti-A2/RA33 autoantibodies nor with MHC genes, although more than 60% of the responsive patients carried the shared epitope. Results obtained with recombinant fragments indicated a major T cell epitope to be located in the N-terminal first RNA binding domain of the protein. Anti-A2/RA33 specific TCC (n = 16) derived from RA patients were almost exclusively CD4?, whereas only 7 of 12 TCC derived from controls showed this phenotype, and secreted high amounts of IFNg upon antigen stimulation as did all TCC derived from controls. Proliferative responses to filaggrin in either form were seen in only 25% of the RA patients tested and did not differ from those observed in the control group indicating that filaggrin-reactive T cells do presumably not drive the autoantibody response to citrullinated antigens. Taken together, a Th1 type autoimmune response to hnRNP-A2/RA33 was commonly observed in RA patients suggesting this nuclear protein to constitute a major T cell autoantigen which might be fuelling one of the pathological autoimmune reactions that drive the destructive processes effective in RA.

Rheumatoid arthritis (RA) is the most common immune-mediated arthritis. Anti-citrullinated peptide antibodies (ACPA) are highly specific to RA and assayed with the commercial CCP2 assay. Genetic drivers of RA within the MHC are different for CCP2-positive and -negative subsets of RA, particularly at HLA-DRB1. However, aspartic acid at amino acid position 9 in HLA-B (Bpos-9) increases risk to both RA subsets. Here we explore how individual serologies associated with RA drive associations within the MHC. To define MHC differences for specific ACPA serologies, we quantified a total of 19 separate ACPAs in RA-affected case subjects from four cohorts (n = 6,805). We found a cluster of tightly co-occurring antibodies (canonical serologies, containing CCP2), along with several independently expressed antibodies (non-canonical serologies). After imputing HLA variants into 6,805 case subjects and 13,467 control subjects, we tested associations between the HLA region and RA subgroups based on the presence of canonical and/or non-canonical serologies. We examined CCP2(+) and CCP2(−) RA-affected case subjects separately. In CCP2(−) RA, we observed that the association between CCP2(−) RA and Bpos-9 was derived from individuals who were positive for non-canonical serologies (omnibus_p = 9.2 × 10−17). Similarly, we observed in CCP2(+) RA that associations between subsets of CCP2(+) RA and Bpos-9 were negatively correlated with the number of positive canonical serologies (p = 0.0096). These findings suggest unique genetic characteristics underlying fine-specific ACPAs, suggesting that RA may be further subdivided beyond simply seropositive and seronegative. A correction has been published: https://doi.org/10.1016/j.ajhg.2019.09.018