Vijay Joshua

Monoclonal IgG antibodies generated from joint-derived B cells of RA patients have a strong bias toward citrullinated autoantigen recognition

Synovial IgG-expressing B cells from patients with rheumatoid arthritis show specificity for citrullinated autoantigens.

Structural Changes and Antibody Enrichment in the Lungs Are Early Features of Anti–Citrullinated Protein Antibody–Positive Rheumatoid Arthritis

It has been suggested that immunologic events in the lungs may be involved in triggering immunity, in particular production of anti–citrullinated protein antibodies (ACPAs) during early phases of rheumatoid arthritis (RA). The aim of this study was to investigate the structural and immunologic features of the lungs in incident cases of early RA in relation to ACPA presence and smoking status.

Identification of a novel chemokine-dependent molecular mechanism underlying rheumatoid arthritis-associated autoantibody-mediated bone loss

Objectives Rheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide antibodies (ACPAs) appear before disease onset and are associated with bone destruction. We aimed to dissect the role of ACPAs in osteoclast (OC) activation and to identify key cellular mediators in this process. Methods Polyclonal ACPA were isolated from the synovial fluid (SF) and peripheral blood of patients with RA. Monoclonal ACPAs were isolated from single SF B-cells of patients with RA. OCs were developed from blood cell precursors with or without ACPAs. We analysed expression of citrullinated targets and peptidylarginine deiminases (PAD) enzymes by immunohistochemistry and cell supernatants by cytometric bead array. The effect of an anti-interleukin (IL)-8 neutralising antibody and a pan-PAD inhibitor was tested in the OC cultures. Monoclonal ACPAs were injected into mice and bone structure was analysed by micro-CT before and after CXCR1/2 blocking with reparixin. Results Protein citrullination by PADs is essential for OC differentiation. Polyclonal ACPAs enhance OC differentiation through a PAD-dependent IL-8-mediated autocrine loop that is completely abolished by IL-8 neutralisation. Some, but not all, human monoclonal ACPAs derived from single SF B-cells of patients with RA and exhibiting distinct epitope specificities promote OC differentiation in cell cultures. Transfer of the monoclonal ACPAs into mice induced bone loss that was completely reversed by the IL-8 antagonist reparixin. Conclusions We provide novel insights into the key role of citrullination and PAD enzymes during OC differentiation and ACPA-induced OC activation. Our findings suggest that IL8-dependent OC activation may constitute an early event in the initiation of the joint specific inflammation in ACPA-positive RA.

Shared immunological targets in the lungs and joints of patients with rheumatoid arthritis: identification and validation

Objectives Immunological events in the lungs might trigger production of anti-citrullinated protein antibodies during early rheumatoid arthritis (RA). We investigated the presence of shared immunological citrullinated targets in joints and lungs of patients with RA. Patients and methods Proteins extracted from bronchial (n=6) and synovial (n=7) biopsy specimens from patients with RA were investigated by mass spectrometry-based proteomics. One candidate peptide was synthesised and used to investigate by ELISA the presence of antibodies in patients with RA (n=393), healthy controls (n=152) and disease controls (n=236). HLA-DRB1 shared epitope (SE) alleles were detected in patients with RA. Results Ten citrullinated peptides belonging to seven proteins were identified, with two peptides shared between the synovial and bronchial biopsy samples. Further analysis, using accurate mass and retention time, enabled detection of eight citrullinated peptides in synovial and seven in bronchial biopsy specimens, with five peptides shared between the synovial and bronchial biopsy specimens. Two citrullinated vimentin (cit-vim) peptides were detected in the majority of synovial and lung tissues. Antibodies to a synthesised cit-vim peptide candidate (covering both cit-vim peptides identified in vivo) were present in 1.8% of healthy controls, 15% of patients with RA, and 3.4% of disease controls. Antibodies to cit-vim peptide were associated with the presence of the SE alleles in RA. Conclusions Identical citrullinated peptides are present in bronchial and synovial tissues, which may be used as immunological targets for antibodies of patients with RA. The data provide further support for a link between lungs and joints in RA and identify potential targets for immunity that may mediate this link.

Mechanisms involved in triggering rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory syndrome with a strong autoimmune component. The autoantigens in RA are neither tissue nor organ‐specific, but comprise a broad collection of post‐translational modified proteins, such as citrullinated proteins. These modifications are likely to be triggered by innate stimuli. In genetically susceptible hosts, they can lead to a more substantiated secondary autoimmune reaction targeting the joints and precipitating the clinical onset of RA. Both innate and adaptive mechanisms will then closely interplay to promote chronic joint inflammation in the several absence of appropriate treatment. This scenario, is shared with other autoimmune diseases where potentially pathogenic immune responses are present already before disease onset. Better understanding of these processes will allow both earlier diagnosis of RA and identification of those healthy individuals that are at risk of developing disease, opening possibilities for disease prevention. In this review, we discuss the iterative processes of innate and adaptive immunity responsible for the (longitudinal) development of immune reactions that may contribute to the development of RA.

The lung microbiota in early rheumatoid arthritis and autoimmunity

BackgroundAirway abnormalities and lung tissue citrullination are found in both rheumatoid arthritis (RA) patients and individuals at-risk for disease development. This suggests the possibility that the lung could be a site of autoimmunity generation in RA, perhaps in response to microbiota changes. We therefore sought to test whether the RA lung microbiome contains distinct taxonomic features associated with local and/or systemic autoimmunity.Methods16S rRNA gene high-throughput sequencing was utilized to compare the bacterial community composition of bronchoalveolar lavage fluid (BAL) in patients with early, disease-modifying anti-rheumatic drugs (DMARD)-naïve RA, patients with lung sarcoidosis, and healthy control subjects. Samples were further assessed for the presence and levels of anti-citrullinated peptide antibodies (including fine specificities) in both BAL and serum.ResultsThe BAL microbiota of RA patients was significantly less diverse and abundant when compared to healthy controls, but similar to sarcoidosis patients. This distal airway dysbiosis was attributed to the reduced presence of several genus (i.e., Actynomyces and Burkhordelia) as well as reported periodontopathic taxa, including Treponema, Prevotella, and Porphyromonas. While multiple clades correlated with local and systemic levels of autoantibodies, the genus Pseudonocardia and various related OTUs were the only taxa overrepresented in RA BAL and correlated with higher disease activity and erosions.ConclusionsDistal airway dysbiosis is present in untreated early RA and similar to that detected in sarcoidosis lung inflammation. This community perturbation, which correlates with local and systemic autoimmune/inflammatory changes, may potentially drive initiation of RA in a proportion of cases.

Serum RANKL levels associate with anti- citrullinated protein antibodies in early untreated rheumatoid arthritis and are modulated following methotrexate

IntroductionReceptor activator of nuclear factor kappa B ligand (RANKL) is a key regulator of bone metabolism. Anti-citrullinated protein antibodies (ACPA) have been suggested to cause bone destruction by osteoclast activation. We investigated the relationship between RANKL and ACPA in patients with early untreated rheumatoid arthritis (RA).MethodsPatients with newly diagnosed untreated RA (n = 183) were analyzed at baseline and 3 months after initiating methotrexate (MTX) treatment. Serum RANKL (total RANKL), ACPA (anti-CCP2) and ACPA specificities (anti-citrullinated (cit)-vimentin, anti-cit-enolase and anti-cit-fibrinogen) were determined by enzyme-linked immunosorbent assay (ELISA). Synovial RANKL expression was evaluated by immunohistochemistry in a small group of patients (n = 15). The relationship between anti-cit-vim antibodies and bone destruction was further validated in 1116 RA patients included in the EIRA cohort. Pearson’s chi-square test, Wilcoxon rank sum test, Wilcoxon signed rank test and linear regression models were used.ResultsSerum RANKL concentration was significantly higher (p <0.05) in ACPA-positive (median: 689 pmol/L, IQR 342–1253) compared with ACPA-negative (median: 159 pmol/L, IQR 96–243) patients and this difference was also seen for synovial RANKL expression. Serum RANKL associated with ACPA (p <0.05) and bone erosions in rheumatoid factor (RF)-negative patients (n = 59). Among ACPA specificites, anti-cit-vimentin (amino acids 60–75) was associated with higher RANKL concentration and higher prevalence of bone erosion (p <0.05). Significant reductions in both serum RANKL and ACPA levels were observed after 3 months of MTX treatment (p <0.05).ConclusionsRANKL was elevated in ACPA-positive and in anti-cit-vimentin-positive patients with early untreated RA and associated with bone erosions. These findings give further support for an early direct pathogenic link between ACPA and bone destruction in RA.

Antibody responses to de novo identified citrullinated fibrinogen peptides in rheumatoid arthritis and visualization of the corresponding B cells

BackgroundAntibodies against citrullinated proteins (ACPA) are common in patients with rheumatoid arthritis (RA). ACPA can appear before disease onset and target many self-antigens. Citrullinated fibrin/fibrinogen represents a classical ACPA target antigen, and mass spectrometry of RA synovial fluid reveals elevated citrullinated (cit) fibrinogen (Fib) peptides compared to non-RA controls. We investigated the extent to which these less-studied peptides represent autoantibody targets and sought to visualize the corresponding cit-Fib-reactive B cells in RA patients.MethodsAn in-house ELISA was established against four cit-Fib α-subunit peptides (cit-Fib α-35; cit-Fib α-216,218; cit-Fib α-263,271 and cit-Fib α-425,426) and serum from patients with established RA (n = 347) and disease controls with psoriatic arthritis (PsA) or ankylosing spondylitis (AS) (n = 236) were analyzed. RA patients were genotyped for HLA-DR alleles, PTPN22 R620W and screened for anti-CCP2 and cit-Fib protein antibodies. The cit-Fib peptides were also used to assemble antigen tetramers to identify cit-Fib-reactive B cells in peripheral blood by flow cytometry.ResultsThe frequencies of autoantibodies against different cit-Fib epitopes in RA patients compared to PsA/AS patients were: cit-Fib α-35 (RA 20%, vs PsA/AS 1%); cit-Fib α-216,218 (13% vs 0.5%); cit-Fib α-263,271 (21% vs 0.5%) and cit-Fib α-425,426 (17% vs 1%). The presence of autoantibodies against these peptides was associated with presence of anti-CCP2 and anti-cit-Fib protein antibodies. No association was found between HLA-DR shared epitope and antibodies to the different cit-Fib peptides. However, association was observed between the PTPN22 risk allele and positivity to cit-Fib α-35 and cit-Fib α-263,271. B cells carrying surface Ig reactive to these cit-Fib peptides were found in RA peripheral blood and these tend to be more common in PTPN22 risk allele carriers.ConclusionsOur data show that several cit-Fib peptides are targeted by autoantibodies in RA, but not in PsA/AS, implicating that these are not due to arthritis but more specific for RA etiology. The RA-associated anti-cit protein response is broad with many parallel immune responses. The association between cit-Fib autoantibodies and the PTPN22 R620W risk allele supports the hypothesis of altered B cell regulation, such as autoreactive B cells evading tolerance checkpoints.

Lung changes detected by high resolution tomography are present in ACPA positive RA patients already at disease onset

Background/purpose The authors have previously shown that smoking increases citrullination in the lungs of healthy smokers and hypothesised that this process is an important step in the early development of rheumatoid arthritis (RA). To test this the authors investigated lung function in a cohort of RA patients with very early disease, already at the time of diagnosis and compared it with an aged, smoke and gender matched cohort of individuals without RA. Methods 82 patients, 56 female and 26 male, median age 60 (range 22–84) with symptom duration less than 1 year at the time of diagnosis and naive to disease-modifying antirheumatic drug treatment were included in the RA cohort. A second cohort of age, smoking and gender matched non-RA individuals (n=43) were investigated with an identical protocol. Lung function (x-ray, high-resolution CT (HRCT) and dynamic spirometry) was tested in both RA and controls at baseline and repeated after 6 months in the RA group. In a subgroup of patients bronchoscopy was performed at inclusion (n=18) and 6 months later (n=11) and bronchoalveolar lavage samples as well as mucosal large bronchial biopsies were retrieved. Presence of peptydilamino deiminase (PAD) enzymes were evaluated by immunohistochemistry. Results 63% of the patients were anticitrullinated protein antibodies (ACPA) positive, 26% were current smokers and 22% have reported a previous lung pathology. All patients had active disease with a mean disease activity score of 5.5±0.1 at inclusion that decreased significantly to 3.3±0.2 after 6 months. A majority of the patients (55%) had any type of changes on HRCT either focal or diffuse. Lung changes of any type were more frequent in RA as compared to non-RA individuals (35%) with a higher prevalence of lung fibrosis (15/82 in the RA cohort and 0/43 in the control cohort). In the RA cohort one patient (1.2%) was diagnosed with lung cancer following HRCT screening while none in the control cohort. ACPA and rheumatoid factor but not smoking status associated with the presence of changes on HRCT. Immunohistochemistry demonstrated a significant increase in expression of both PAD2 and PAD4 in the lungs of current smokers independent of the ACPA status with no significant changes following 6 months of treatment. Conclusion Presence of ACPA associates with early lung HRCT changes in RA. The authors suggest that smoking (and other yet unidentified factors) promotes site specific citrullination (such as in lungs) leading to generation of ACPA immunity and lung changes very early in the disease process.

Role of the lung in individuals at risk of rheumatoid arthritis

Antibody-positive (seropositive) rheumatoid arthritis (RA) is a complex multiphasic disease developing in genetically susceptible individuals following environmental challenges (such as smoking). RA-associated autoantibodies can develop several years before any clinical signs of joint inflammation, suggesting that triggering of this autoimmunity occurs outside the joints. Epidemiological, clinical, and molecular studies in seropositive individuals at risk for developing RA as well as in early untreated RA suggest a potential role for mucosal sites (especially lung mucosa) as RA-associated autoimmunity trigger sites. This chapter summarizes clinical and molecular studies supporting the lung as a central site for autoimmunity initiation in RA.