Studies on the role of anti-citrullinated protein antibodies in rheumatoid arthritis

Anti-citrullinated protein antibodies (ACPA) are highly specific for rheumatoid arthritis (RA) and present in about two thirds of all patients at diagnosis. They can be detected already before disease onset and have direct pathogenic effect mediated partly through the Fc (fragment crystalizable) portion with attached Fc-glycan structure. On these grounds we aimed to further characterize ACPA’s role in the pathogenesis of RA both as a risk factor and a disease biomarker. To this end we investigated ACPA occurrence in a population-derived twin cohort and ACPA in a cohort of early-untreated RA patients in relation to disease outcomes. First we screened a large population-derived twin cohort (N= 12,590; median age 64, range 48-93 years) for occurrence of ACPA using an ACPA test used in clinical routine: the Anti-CCP2 (IgG) test. Through linking the twin cohort with the Swedish National Patient Register we identified ACPA-positive individuals without RA (N=226) and ACPA-positive patients with RA (N=124). ACPA-positive individuals without RA had lower ACPA concentration and fewer different ACPA reactivities as compared to patients with ACPA-positive RA. Heritability estimates for having ACPA with or without RA were generally lower than expected (10% (95% CI: 0-43) for ACPA without RA; 23% (95% CI: 0-45) for ACPA; and 41% (95% CI: 0-74) for ACPA-positive RA). Heavy smoking and HLA-SE associated with ACPA occurrence with and without RA. Environmental factors (including smoking) appeared to be more important than genetic in determining which individuals develop ACPA, while genetic factors (and in particular HLA-SE) had a relatively larger impact in determining which ACPA-positive individuals that will ultimately develop arthritis. We also confirmed that presence of ACPA and especially high titers of ACPA have a high diagnostic accuracy for RA in a population based setting. Following this, we then screened a cohort of early-untreated RA patients (N=183) for occurrence of ACPA using either the Anti-CCP2 test or ELISA for detection of reactivities against specific citrullinated peptides. We demonstrated that ACPA (and especially anti-citrullinated-vimentin antibodies) associated with markers of bone loss (as measured by ELISA detection of serum RANKL and/or presence of bone erosions on radiographs of hands and feet). Treatment with methotrexate (MTX) significantly lowered both ACPA and RANKL serum levels. In a subgroup of these patients (N=59) we investigated the Fc-glycosylation patterns of serum IgG in relation to disease outcome further. A general low abundance of galactosylated glycans, partially restored by MTX treatment, was observed in the serum of early-untreated RA samples. This was more evident among future non-responders as compared to responders to MTX treatment. The galactosylation status of the IgG-Fc had good predictive value for MTX response. In conclusion we showed that environmental as well as genetic factors are important for ACPA occurrence, which in turn has a high diagnostic accuracy for RA. In early-untreated RA, ACPA associate with bone loss and is modulated by methotrexate treatment. Further, Fc-glycosylation patterns of antibodies are generally altered in early-untreated RA and might serve as a predictive factor for therapeutic response

T cells are influenced by a long non-coding RNA in the autoimmune associated PTPN2 locus

Non-coding SNPs in the protein tyrosine phosphatase non-receptor type 2 (PTPN2) locus have been linked with several autoimmune diseases, including rheumatoid arthritis, type I diabetes, and inflammatory bowel disease. However, the functional consequences of these SNPs are poorly characterized. Herein, we show in blood cells that SNPs in the PTPN2 locus are highly correlated with DNA methylation levels at four CpG sites downstream of PTPN2 and expression levels of the long non-coding RNA (IncRNA) LINC01882 downstream of these CpG sites. We observed that LINC01882 is mainly expressed in T cells and that anti-CD3/CD28 activated naive CD4(+) T cells downregulate the expression of LINC01882. RNA sequencing analysis of LINC01882 knockdown in Jurkat T cells, using a combination of antisense oligo-nucleotides and RNA interference, revealed the upregulation of the transcription factor ZEB1 and kinase MAP2K4, both involved in IL-2 regulation. Overall, our data suggests the involvement of LINC01882 in T cell activation and hints towards an auxiliary role of these non-coding SNPs in autoimmunity associated with the PTPN2 locus.

Association between number and type of different ACPA fine specificities with lung abnormalities in early, untreated rheumatoid arthritis

International audienceBackground Rheumatoid arthritis (RA)-associated anticitrullinated protein/peptide antibodies (ACPA) might originate at mucosal sites such as the lungs. We aimed to examine the relationship between the ACPA repertoire and lung abnormalities on high-resolution CT (HRCT) in patients with earlyuntreated RA. Methods 106 patients with newly diagnosed untreated RA were examined with HRCT of the lungs. Blood samples were analysed for presence of rheumatoid factor (RF) and ACPA using either a CCP2 detection kit or an immunochip containing 10 different citrullinated peptides. Association between HRCT findings and the antibody repertoire was assessed by logistic regression analysis. Results The number (%) of patients with HRCT abnormalities was 58 (54.7%) for parenchymal abnormalities and 68 (64.2%) for airway abnormalities. CCP2 IgG, RF IgA and antibodies against citrullinated fibrinogen were associated with the presence of parenchymal lung abnormalities. Interestingly, a high number of ACPA fine specificities gave a high risk of having parenchymal lung abnormalities at the time of RA diagnosis. No significant signals were identified between ACPA specificities and risk for airway abnormalities. Conclusions The presence of RF and ACPAs (especially against citrullinated fibrinogen peptides) as well as high number of ACPAs fine specificities are associated with parenchymal lung abnormalities in patients with early, untreated RA. This provides further support for an important pathogenic link between the lung and systemic autoimmunity, contributing to RA development

The role of anti-citrullinated protein antibody reactivities in an inception cohort of patients with rheumatoid arthritis receiving treat-to-target therapy

Background: Anti-citrullinated protein antibody (ACPA) reactivities precede clinical onset of rheumatoid arthritis (RA), and it has been suggested that ACPA reactivities towards distinct target proteins may be associated with differences in RA phenotypes. We aimed to assess the prevalence of baseline ACPA reactivities in an inception cohort of patients with early RA, and to investigate their associations with disease activity, treatment response, ultrasound findings and radiographic damage. Methods: Disease-modifying antirheumatic drug (DMARD)-naïve patients with early RA, classified according to the 2010 American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) criteria, were included in the ARCTIC trial and assessed in the present analysis. During follow up, patients were monitored frequently and treatment was adjusted according to a predetermined protocol, starting with methotrexate monotherapy with prednisolone bridging. Analysis of 16 different ACPA reactivities targeting citrullinated peptides from fibrinogen, alpha-1 enolase, vimentin, filaggrin and histone was performed using a multiplex chip-based assay. Samples from 0, 3, 12 and 24 months were analysed. Controls were blood donors with similar characteristics to the patients (age, gender, smoking status). Results: A total of 217 patients and 94 controls were included. Median [25, 75 percentile] number of ACPA reactivities in all patients was 9 [4, 12], and were most prevalent in anti-cyclic citrullinated peptide /rheumatoid factor-positive patients 10 [7, 12]. Disease activity measures and ultrasound scores at baseline were lower in ACPA reactivity-positive compared to ACPA reactivity-negative patients. ACPA reactivity levels decreased after 3 months of DMARD treatment, most pronounced for fibrinogenβ 60–74 to 62% of baseline antibody level, with least change in filaggrin 307–324 to 81% of baseline antibody level, both p < 0.001. However, outcomes in disease activity measures, ultrasound and radiographic scores after 12 and 24 months were not associated with baseline levels or changes in ACPA reactivity levels and/or seroreversion after 3 months. Conclusions: The clinical relevance of analysing ACPA reactivities in intensively treated and closely monitored early RA was limited, with no apparent associations with disease activity, prediction of treatment response or radiographic progression. Further studies in larger patient materials are needed to understand the role of ACPA reactivities in patients with RA classified according to the 2010 ACR/EULAR criteria and treated according to modern treatment strategies

Anticitrullinated protein antibodies facilitate migration of synovial tissue-derived fibroblasts

OBJECTIVES Rheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide antibodies (ACPAs) might contribute to bone loss and arthralgia before the onset of joint inflammation. We aimed to dissect additional mechanisms by which ACPAs might contribute to development of joint pathology. METHODS Fibroblast-like synoviocytes (FLS) were isolated from the synovial membrane of patients with RA. The FLS cultures were stimulated with polyclonal ACPAs (anti-CCP-2 antibodies) purified from the peripheral blood of patients with RA or with monoclonal ACPAs derived from single synovial fluid B cells. We analysed how ACPAs modulate FLS by measuring cell adhesion and mobility as well as cytokine production. Expression of protein arginine deiminase (PAD) enzymes and protein citrullination were analysed by immunofluorescence, and signal transduction was studied using immunoblotting. RESULTS Challenge of FLS by starvation-induced stress or by exposure to the chemokine interleukin-8 was essential to sensitise the cells to ACPAs. These challenges led to an increased PAD expression and protein citrullination and an ACPA-mediated induction of FLS migration through a mechanism involving phosphoinositide 3-kinase activation. Inhibition of the PAD enzymes or competition with soluble citrullinated proteins or peptides completely abolished the ACPA-induced FLS migration. Different monoclonal ACPAs triggered distinct cellular effects in either fibroblasts or osteoclasts, suggesting unique roles for individual ACPA clones in disease pathogenesis. CONCLUSION We propose that transient synovial insults in the presence of a certain pre-existing ACPA repertoire might result in an ACPA-mediated increase of FLS migration