Autoreactive B cells against malondialdehyde-induced protein cross-links are present in the joint, lung, and bone marrow of rheumatoid arthritis patients.

Autoantibodies to malondialdehyde (MDA) proteins constitute a subset of anti-modified protein autoantibodies in rheumatoid arthritis (RA), which is distinct from citrulline reactivity. Serum anti-MDA IgG levels are commonly elevated in RA and correlate with disease activity, CRP, IL6, and TNF-α. MDA is an oxidation-associated reactive aldehyde that together with acetaldehyde mediates formation of various immunogenic amino acid adducts including linear MDA-lysine, fluorescent malondialdehyde acetaldehyde (MAA)-lysine, and intramolecular cross-linking. We used single-cell cloning, generation of recombinant antibodies (n = 356 from 25 donors), and antigen-screening to investigate the presence of class-switched MDA/MAA+ B cells in RA synovium, bone marrow, and bronchoalveolar lavage. Anti-MDA/MAA+ B cells were found in bone marrow plasma cells of late disease and in the lung of both early disease and risk-individuals and in different B cell subsets (memory, double negative B cells). These were compared with previously identified anti-MDA/MAA from synovial memory and plasma cells. Seven out of eight clones carried somatic hypermutations and all bound MDA/MAA-lysine independently of protein backbone. However, clones with somatic hypermutations targeted MAA cross-linked structures rather than MDA- or MAA-hapten, while the germline-encoded synovial clone instead bound linear MDA-lysine in proteins and peptides. Binding patterns were maintained in germline converted clones. Affinity purification of polyclonal anti-MDA/MAA from patient serum revealed higher proportion of anti-MAA versus anti-MDA compared to healthy controls. In conclusion, IgG anti-MDA/MAA show distinct targeting of different molecular structures. Anti-MAA IgG has been shown to promote bone loss and osteoclastogenesis in vivo and may contribute to RA pathogenesis.

Identification of early risk factors for anti-citrullinated-protein-antibody positive rheumatoid arthritis-a prospective cohort study.

OBJECTIVE: Individuals positive for anti-cyclic-peptide-antibodies (anti-CCP) and musculoskeletal complaints (MSK-C) are at risk for developing rheumatoid arthritis (RA). In this study we aimed to investigate factors involved in arthritis progression. METHODS: Anti-CCP2-positive individuals with MSK-C referred to a rheumatologist were recruited. Individuals lacked arthritis at clinical and ultrasound examination and were followed for ≥three years or until clinical arthritis diagnosis. Blood samples from inclusion were analyzed for; nine anti-citrullinated-protein-antibody (ACPA) reactivities (citrullinated α-1-enolase, fibrinogen, filaggrin, histone, vimentin and tenascin peptides); 92 inflammation-associated proteins; and HLA-shared epitope alleles. Cox regression was applied to the data to identify independent predictors in a model. RESULTS: 267 individuals were included with median follow up of 49 months (IQR: 22-60). 101 (38%) developed arthritis after median 14 months (IQR: 6-27). The analysis identified that presence of at least one ACPA reactivity (HR 8.0, 95% CI 2.9-22), ultrasound detected tenosynovitis (HR 3.4, 95% CI 2.0-6.0), IL6 levels (HR 1.5, 95% CI 1.2-1.8) and IL15-Rα levels (HR 0.6, 95% CI 0.4-0.9) are significant independent predictors for arthritis progression in a prediction model (Harrell's C 0.76 [SE 0.02], AUC 0.82 [95% CI 0.76-0.89], cross-validated AUC 0.70 [95% CI 0.56-0.85]). CONCLUSION: We propose a high-Risk-RA phase characterized by presence of ACPA reactivity, tenosynovitis, IL6, and IL15-Rα and suggest that these factors need to be further investigated for their biological effects and clinical values, to identify individuals at particular low risk and high risk for arthritis progression.

The human bone marrow plasma cell compartment in rheumatoid arthritis - Clonal relationships and anti-citrulline autoantibody producing cells.

A majority of circulating IgG is produced by plasma cells residing in the bone marrow (BM). Long-lived BM plasma cells constitute our humoral immune memory and are essential for infection-specific immunity. They may also provide a reservoir of potentially pathogenic autoantibodies, including rheumatoid arthritis (RA)-associated anti-citrullinated protein autoantibodies (ACPA). Here we investigated paired human BM plasma cell and peripheral blood (PB) B-cell repertoires in seropositive RA, four ACPA+ RA patients and one ACPA- using two different single-cell approaches, flow cytometry sorting, and transcriptomics, followed by recombinant antibody generation. Immunoglobulin (Ig) analysis of >900 paired heavy-light chains from BM plasma cells identified by either surface CD138 expression or transcriptome profiles (including gene expression of MZB1, JCHAIN and XBP1) demonstrated differences in IgG/A repertoires and N-linked glycosylation between patients. For three patients, we identified clonotypes shared between BM plasma cells and PB memory B cells. Notably, four individuals displayed plasma cells with identical heavy chains but different light chains, which may indicate receptor revision or clonal convergence. ACPA-producing BM plasma cells were identified in two ACPA+ patients. Three of 44 recombinantly expressed monoclonal antibodies from ACPA+ RA BM plasma cells were CCP2+, specifically binding to citrullinated peptides. Out of these, two clones reacted with citrullinated histone-4 and activated neutrophils. In conclusion, single-cell investigation of B-cell repertoires in RA bone marrow provided new understanding of human plasma cells clonal relationships and demonstrated pathogenically relevant disease-associated autoantibody expression in long-lived plasma cells.

Presence of anti-modified protein antibodies in idiopathic pulmonary fibrosis.

BACKGROUND AND OBJECTIVE: Studies of autoimmunity and anti-citrullinated protein antibodies (ACPA) in idiopathic pulmonary fibrosis (IPF) have been confined to investigations of anti-cyclic citrullinated peptide (anti-CCP) antibodies which utilize synthetic peptides as surrogate markers for in vivo citrullinated antigens. We studied immune activation by analysing the prevalence of in vivo anti-modified protein antibodies (AMPA) in IPF. METHODS: We included patients with incident and prevalent IPF (N = 120), sex and smoking-matched healthy controls (HC) (N = 120) and patients with RA (N = 104). Serum (median time: 11 months [Q1-Q3: 1-28 months] from diagnosis) was analysed for presence of antibodies towards native and posttranslational modified (citrullinated [Cit, N = 25]; acetylated [Acet, N = 4] and homocitrullinated [Carb, N = 1]) peptides derived from tenascin (TNC, N = 9), fibrinogen (Fib, N = 11), filaggrin (Fil, N = 5), histone (N = 8), cathelicidin (LL37, N = 4) and vimentin (N = 5) using a custom-made peptide microarray. RESULTS: AMPA were more frequent and in increased levels in IPF than in HC (44% vs. 27%, p < 0.01), but less than in RA (44% vs. 79%, p < 0.01). We specifically observed AMPA in IPF towards certain citrullinated, acetylated and carbamylated peptides versus HC: tenascin (Cit(2033) -TNC2025-2040 ; Cit(2197) -TNC2177-2200 ; Cit(2198) -TNC2177-2200 ), fibrinogen (Cit(38,42) -Fibα36-50 ; Cit(72) -Fibß60-74 ) and filaggrin (Acet-Fil307-324 , Carb-Fil307-324 ). No differences in survival (p = 0.13) or disease progression (p = 0.19) between individuals with or without AMPA was observed in IPF. However, patients with incident IPF had better survival if AMPA were present (p = 0.009). CONCLUSION: A significant proportion of IPF patients present with specific AMPA in serum. Our results suggest autoimmunity as a possible characteristic for a subgroup of IPF that may affect disease outcome.

Towards prevention of autoimmune diseases: The example of rheumatoid arthritis.

Prevention is the ultimate aim for clinicians and scientists concerned with severe diseases, like many immune-mediated conditions. Here, we describe recent progress in the understanding of etiology and molecular pathogenesis of rheumatoid arthritis (RA), which make this disease a potential prototype for prevention that may include both public health measures and targeted and personalized approaches that we call "personalized prevention." Critical components of this knowledge are (i) better understanding of the dynamics of the RA-associated autoimmunity that may begin many years before onset of joint inflammation; (ii) insights into how this immunity may be triggered at mucosal surfaces after distinct environmental challenges; (iii) better understanding of which features of the pre-existing immunity may cause symptoms that precede joint inflammation and predict a high risk for imminent arthritis development; and (iv) how molecular events occurring before onset of inflammation might be targeted by existing or future therapies, ultimately by specific targeting of Major histocompatibility complex (MHC) class II restricted and RA-specific immunity. Our main conclusion is that studies and interventions in the phase of autoimmunity preceding RA offer new opportunities to prevent the disease and thereby also understand the molecular pathogenesis of its different variants.

Multi-HLA class II tetramer analyses of citrulline-reactive T cells and early treatment response in rheumatoid arthritis.

BACKGROUND: HLA class II tetramers can be used for ex vivo enumeration and phenotypic characterisation of antigen-specific CD4+ T cells. They are increasingly applied in settings like allergy, vaccination and autoimmune diseases. Rheumatoid arthritis (RA) is a chronic autoimmune disorder for which many autoantigens have been described. RESULTS: Using multi-parameter flow cytometry, we developed a multi-HLA class II tetramer approach to simultaneously study several antigen specificities in RA patient samples. We focused on previously described citrullinated HLA-DRB1*04:01-restricted T cell epitopes from α-enolase, fibrinogen-ß, vimentin as well as cartilage intermediate layer protein (CILP). First, we examined inter-assay variability and the sensitivity of the assay in peripheral blood from healthy donors (n = 7). Next, we confirmed the robustness and sensitivity in a cohort of RA patients with repeat blood draws (n = 14). We then applied our method in two different settings. We assessed lymphoid tissue from seropositive arthralgia (n = 5) and early RA patients (n = 5) and could demonstrate autoreactive T cells in individuals at risk of developing RA. Lastly, we studied peripheral blood from early RA patients (n = 10) and found that the group of patients achieving minimum disease activity (DAS28 < 2.6) at 6 months follow-up displayed a decrease in the frequency of citrulline-specific T cells. CONCLUSIONS: Our study demonstrates the development of a sensitive tetramer panel allowing simultaneous characterisation of antigen-specific T cells in ex vivo patient samples including RA 'at risk' subjects. This multi-tetramer approach can be useful for longitudinal immune-monitoring in any disease with known HLA-restriction element and several candidate antigens.

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.

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 (lncRNA) LINC01882 downstream of these CpG sites. We observed that LINC01882 is mainly expressed in T cells and that anti-CD3/CD28 activated naïve CD4+ T cells downregulate the expression of LINC01882. RNA sequencing analysis of LINC01882 knockdown in Jurkat T cells, using a combination of antisense oligonucleotides 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.

How well do ACPA discriminate and predict RA in the general population: a study based on 12†590 population-representative Swedish twins.

OBJECTIVE: Anti-citrullinated protein antibodies (ACPA) are highly specific for rheumatoid arthritis (RA), but the diagnostic accuracy of ACPA in the general population has not been thoroughly assessed. We aimed to assess the diagnostic accuracy of ACPA for RA in the general population and to further characterise the citrullinated peptide recognition pattern. METHODS: Serum samples from a large population-representative twin cohort consisting of 12 590 individuals were analysed for the presence of ACPA using anti-CCP2 ELISA. All ACPA-positive samples were further tested on ELISAs for four peptide-specific ACPA. RA cases were identified by linkage to the Swedish National Patient Register at inclusion and after a median follow-up of 37 months (IQR 31-49). RESULTS: 350 out of 12 590 individuals had a positive anti-CCP2 test, measuring ACPA. Of these, 103 had an RA diagnosis at the time of blood donation and inclusion. During a median follow-up of 3 years, an additional 21 of the remaining 247 ACPA-positive individuals developed RA. Overall, a positive anti-CCP2 test had a positive predictive value of 29% for prevalent RA at inclusion (negative predictive value of 99.6%). High titres (>3× cut-off) of anti-CCP2 increased the positive predictive value to 48% (negative predictive value of 99.5%). ACPA-positive individuals without RA had lower anti-CCP2 titres and fewer peptide-specific ACPA than ACPA-positive patients with RA and higher C reactive protein levels than ACPA-negative individuals without RA. CONCLUSION: Presence of ACPA and especially high titres of anti-CCP2 have a high diagnostic accuracy for an RA diagnosis in a population setting.

Integration of known DNA, RNA and protein biomarkers provides prediction of anti-TNF response in rheumatoid arthritis: results from the COMBINE study.

OBJECTIVE: In rheumatoid arthritis (RA) several recent efforts have sought to discover means of predicting which patients would benefit from treatment. However, results have been discrepant with few successful replications. Our objective was to build a biobank with DNA, RNA and protein measurements to test the claim that the current state-of-the-art precision medicine will benefit RA patients. METHODS: We collected 451 blood samples from 61 healthy individuals and 185 RA patients initiating treatment, before treatment initiation and at a 3 month follow-up time. All samples were subjected to high-throughput RNA sequencing, DNA genotyping, extensive proteomics and flow cytometry measurements, as well as comprehensive clinical phenotyping. Literature review identified 2 proteins, 52 single-nucleotide polymorphisms (SNPs) and 72 gene-expression biomarkers that had previously been proposed as predictors of TNF inhibitor response (∆DAS28-CRP). RESULTS: From these published TNFi biomarkers we found that 2 protein, 2 SNP and 8 mRNA biomarkers could be replicated in the 59 TNF initiating patients. Combining these replicated biomarkers into a single signature we found that we could explain 51% of the variation in ∆DAS28-CRP. This corresponds to a sensitivity of 0.73 and specificity of 0.78 for the prediction of three month ∆DAS28-CRP better than -1.2. CONCLUSIONS: The COMBINE biobank is currently the largest collection of multi-omics data from RA patients with high potential for discovery and replication. Taking advantage of this we surveyed the current state-of-the-art of drug-response stratification in RA, and identified a small set of previously published biomarkers available in peripheral blood which predicts clinical response to TNF blockade in this independent cohort.

Protective effect of HLA-DRB1*13 alleles during specific phases in the development of ACPA-positive RA.

OBJECTIVE: Human leucocyte antigen (HLA)-DRB1*13 alleles are associated with protection from anticitrullinated protein antibody (ACPA)-positive rheumatoid arthritis (RA). It is, however, unknown at which phase of disease development (seroconversion, ACPA maturation, disease onset or outcome) these alleles are most important. We therefore examined the effect of HLA-DRB1*13 on: ACPA presence (systemic autoimmunity associated with RA) in individuals with and without RA, on ACPA characteristics and on clinical outcome measures. METHODS: The effect of HLA-DRB1*13 on ACPA presence in subjects with or without RA (non-RA) was assessed in the Swedish twin registry (n=10 748). ACPA characteristics were studied in patients with ACPA-positive RA from the Swedish Epidemiological Investigation of RA (EIRA, n=1224) and the Dutch Leiden Early Arthritis Clinic (EAC, n=441). Disease activity at inclusion and disease outcome (disease-modifying antirheumatic drugs (DMARD)-free sustained remission and radiographic progression) was assessed in patients with RA from the EAC. RESULTS: HLA-DRB1*13 is associated with protection from ACPA-positive RA (prevalence 16% vs 28% in ACPA-negative non-RA), but not with significant protection from ACPA in individuals without RA (prevalence: 22%, p value 0.09). HLA-DRB1*13 is associated with lower ACPA-levels (EIRA: 447 U/ml versus 691 U/ml, p value= 0.0002) and decreased citrullinated epitope recognition (EIRA: p<0.0001). No association between HLA-DRB1*13 and disease activity or outcome was found. CONCLUSIONS: These data indicate that HLA-DRB1*13 mainly affects the onset of ACPA-positive RA in ACPA-positive non-RA individuals. In RA, HLA-DRB1*13 influences ACPA characteristics but not the disease course. This implies that therapeutic strategies aimed at emulating the HLA-DBR1*13 protective effect may be most effective in ACPA-positive healthy individuals at risk for RA.

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.

Environmental and genetic factors in the development of anticitrullinated protein antibodies (ACPAs) and ACPA-positive rheumatoid arthritis: an epidemiological investigation in twins.

OBJECTIVE: To investigate the role of genetic and environmental factors in the development of anticitrullinated protein antibodies (ACPA) and ACPA-positive rheumatoid arthritis (RA) in a twin cohort. METHODS: A total of 12 590 twins were analysed for the presence of ACPAs (CCP2 ELISA), HLA-DRB1 shared epitope (SE) gene alleles, and exposure to smoking. Twins with established RA were identified in national public care registers. Antibody reactivities against citrullinated and native forms of α-enolase, vimentin, fibrinogen and type II collagen peptides were tested by ELISA in anti-CCP2-positive subjects and their cotwins. Structural equation models and ORs for the development of ACPA and ACPA-positive RA were computed for smokers and SE carriers. RESULTS: A total of 2.8% (350/12 590) of the twins were ACPA positive, and 1.0% (124/12 590) had ACPA-positive RA. Most of the variability in the ACPA status was accounted for by non-shared environmental or stochastic factors (78%, 95% CI 55% to 100%) rather than shared environmental and genetic factors. Analysis of specific risk factors revealed an association between smoking and SE and the presence of ACPAs. Twins with ACPA-positive RA were more frequently SE positive than twins with ACPAs without RA. Reactivities against multiple citrullinated peptides were present in most twins with ACPA-positive RA but in fewer twins with ACPAs without RA. CONCLUSIONS: Environment, lifestyle and stochastic factors may be more important than genetics in determining which individuals develop ACPAs. Genetic factors (particularly SE) may have a relatively larger role in determining which ACPA-positive individuals will ultimately develop arthritis.

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.

Distinct Fluorescence Optical Imaging Patient Clusters Emerge for Seropositive and Seronegative Rheumatoid Arthritis.

OBJECTIVE: To investigate whether digital activity fluorescence optical imaging (FOI) patterns of inflammation can identify distinct rheumatoid arthritis (RA) phenotypes. METHODS: The hands of newly diagnosed patients with RA were evaluated by clinical examination, musculoskeletal ultrasound, and FOI. Inflammation on FOI was defined when capillary leakage and/or fluorophore perfusion was present. The FOI composite image was quantified into a digital disease activity (DACT) score, using novel computerized algorithms. Unsupervised clustering on FOI inflammatory patterns was used to identify subgroups of patients relative to anticyclic citrullinated peptides (ACPA) and/or rheumatoid factor (RF). RESULTS: Of 1326 examined hand joints in 39 patients with RA (72% female; 56% ever-smokers; 54% RF positive and 69% ACPA positive), 400 (30%) showed inflammation by FOI, and 95% (37 of 39) of patients had DACT-FOI scores greater than 1. Unsupervised analysis on FOI patterns revealed two patient clusters, cluster 1 (n = 29) and cluster 2 (n = 10). The proportion of seropositive patients was significantly higher in cluster 1 versus cluster 2 (90%, 26 of 29 vs. 30%, 3 of 10; P < 0.01), whereas C-reactive-protein levels (minimum-maximum) were significantly higher in cluster 2 (20 mg/l [1-102]) versus cluster 1 (2 mg/l [0-119]; P = 0.01). A wider variety and proportion of inflamed joints emerged for patients with RA in cluster 2 versus cluster 1, in which inflammation was more concentrated around the wrists and the right metacarpophalangeal 2 (MCP2), bilateral MCP3, and, to a lesser degree, left MCP2 and proximal interphalangeal joint and tendon regions. Cluster 1 displayed lower mean (±SD) DACT scores compared with cluster 2 (3.6 ± 2.1 vs. 5.4 ± 2.1; P = 0.03). CONCLUSION: FOI-based digital quantification of hand joint inflammation revealed two distinct RA subpopulations with and without ACPA and RF related autoantibodies.

Combination of Two Monoclonal Anti-Citrullinated Protein Antibodies Induced Tenosynovitis, Pain, and Bone Loss in Mice in a Peptidyl Arginine Deiminase-4-Dependent Manner.

OBJECTIVE: The appearance of anti-citrullinated protein antibodies (ACPAs) in the circulation represents a major risk factor for developing rheumatoid arthritis (RA). Patient-derived ACPAs have been shown to induce pain and bone erosion in mice, suggesting an active role in the pathogenicity of RA. We undertook this study to investigate whether ACPAs can induce tenosynovitis, an early sign of RA, in addition to pain and bone loss and whether these symptoms are dependent on peptidyl arginine deiminase 4 (PAD4). METHODS: Monoclonal ACPAs generated from plasma cells of RA patients were transferred to wild-type and PAD4-deficient mice. Pain-like behavior and macroscopic inflammation were monitored for a period of 4 weeks, followed by the analyses of tenosynovitis in the ankle joints using magnetic resonance imaging (MRI) and bone microarchitecture in the tibia using an X-ray microscope. Microscopic changes in the tendon sheath were analyzed in decalcified ankle joint sections. RESULTS: The combination of 2 monoclonal ACPAs (1325:04C03 and 1325:01B09) induced long-lasting pain-like behavior and trabecular bone loss in mice. Although no synovitis was observed macroscopically, we detected tenosynovitis in the ACPA-injected mice by MRI. Microscopic analyses of the joints revealed a cellular hyperplasia and a consequent enlargement of the tendon sheath in the ACPA-treated group. In PAD4-/- mice, the effects of ACPAs on pain-like behavior, tenosynovitis, and bone loss were significantly reduced. CONCLUSION: Monoclonal ACPAs can induce tenosynovitis in addition to pain and bone loss via mechanisms dependent on PAD4-mediated citrullination.

Rheumatoid Arthritis-Specific Autoimmunity in the Lung Before and at the Onset of Disease.

OBJECTIVE: The lung is implicated as a site for breach of tolerance prior to onset of seropositive rheumatoid arthritis (RA). To substantiate this, we investigated lung-resident B cells in bronchoalveolar lavage (BAL) samples from untreated early RA patients and anti-citrullinated protein antibody (ACPA)-positive individuals at risk for developing RA. METHODS: Single B cells (n = 7,680) were phenotyped and isolated from BAL samples from individuals at risk of RA (n = 3) and at RA diagnosis (n = 9). The immunoglobulin variable region transcripts were sequenced and selected for expression as monoclonal antibodies (n = 141). Monoclonal ACPAs were tested for reactivity patterns and binding to neutrophils. RESULTS: Using our single-cell approach, we found significantly increased proportions of B lymphocytes in ACPA+ compared to ACPA- individuals. Memory and double-negative B cells were prominent in all subgroups. Upon antibody re-expression, 7 highly mutated citrulline-autoreactive clones originating from different memory B cell subsets were identified, both in individuals at risk of RA and early RA patients. Lung IgG variable gene transcripts from ACPA+ individuals carried frequent mutation-induced N-linked Fab glycosylation sites (P < 0.001), often in the framework 3 of the variable region. Two of the lung ACPAs bound to activated neutrophils, 1 from an individual at risk of RA and 1 from an early RA patient. CONCLUSION: T cell-driven B cell differentiation resulting in local class switching and somatic hypermutation are evident in lungs before as well as in early stages of ACPA+ RA. Our findings add to the notion of lung mucosa being a site for initiation of citrulline autoimmunity preceding seropositive RA.

Molecular signature of methotrexate response among rheumatoid arthritis patients.

Background: Methotrexate (MTX) is the first line treatment for rheumatoid arthritis (RA), but failure of satisfying treatment response occurs in a significant proportion of patients. Here we present a longitudinal multi-omics study aimed at detecting molecular and cellular processes in peripheral blood associated with a successful methotrexate treatment of rheumatoid arthritis. Methods: Eighty newly diagnosed patients with RA underwent clinical assessment and donated blood before initiation of MTX, and 3 months into treatment. Flow cytometry was used to describe cell types and presence of activation markers in peripheral blood, the expression of 51 proteins was measured in serum or plasma, and RNA sequencing was performed in peripheral blood mononuclear cells (PBMC). Response to treatment after 3 months was determined using the EULAR response criteria. We assessed the changes in biological phenotypes during treatment, and whether these changes differed between responders and non-responders with regression analysis. By using measurements from baseline, we also tried to find biomarkers of future MTX response or, alternatively, to predict MTX response. Results: Among the MTX responders, (Good or Moderate according to EULAR treatment response classification, n = 60, 75%), we observed changes in 29 partly overlapping cell types proportions, levels of 13 proteins and expression of 38 genes during treatment. These changes were in most cases suppressions that were stronger among responders compared to non-responders. Within responders to treatment, we observed a suppression of FOXP3 gene expression, reduction of immunoglobulin gene expression and suppression of genes involved in cell proliferation. The proportion of many HLA-DR expressing T-cell populations were suppressed in all patients irrespective of clinical response, and the proportion of many IL21R+ T-cells were reduced exclusively in non-responders. Using only the baseline measurements we could not detect any biomarkers or prediction models that could predict response to MTX. Conclusion: We conclude that a deep molecular and cellular phenotyping of peripheral blood cells in RA patients treated with methotrexate can reveal previously not recognized differences between responders and non-responders during 3 months of treatment with MTX. This may contribute to the understanding of MTX mode of action and explain non-responsiveness to MTX therapy.