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.

In early rheumatoid arthritis, anticitrullinated peptide antibodies associate with low number of affected joints and rheumatoid factor associates with systemic inflammation.

OBJECTIVES: To investigate how individual rheumatoid arthritis (RA) autoantibodies associate with individual signs and symptoms at the time of RA diagnosis. METHODS: IgA, IgG, IgM rheumatoid factor (RF), antibodies against cyclic citrullinated peptide version 2 (anti-CCP2) and 16 individual antibodies against citrullinated protein (ACPA) reactivities were analysed centrally in baseline sera from 1600 patients with RA classified according to the 1987 American College of Rheumatology (ACR) criteria. These results were related to C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), number of swollen and tender joints (SJC and TJC), 28-joint disease activity scores (DAS28 and DAS28CRP), global disease activity evaluated by the patients and Health Assessment Questionnaire, all obtained at baseline. RESULTS: Individually, all autoantibodies except immunoglobulin G (IgG) RF associated with low SJC and TJC and with high ESR. In IgM RF-negative patients, ACPA associated strictly with low number of swollen and tender joints. This association persisted in multiple regression and stratified analyses where IgM and IgA RF instead associated with inflammation expressed as ESR. Among subjects without any ACPA peptide reactivity, there was no association between RF isotypes and ESR. The effect of RF on ESR increased with the number of ACPA reactivities, especially for IgM RF. In patients fulfilling the 1987 ACR criteria without taking RF into account, associations between IgM RF and high ESR, as well as between ACPA and low joint counts, remained. CONCLUSION: Whereas ACPA associate with low counts of affected joints in early RA, RF associates with elevated measures of systemic inflammation in an ACPA-dependent manner. This latter finding corroborates in vitro models of ACPA and RF in immune complex-induced inflammation. These phenotypic associations are independent of classification criteria.

Identification of circulating autoantibodies to non-modified proteins associated with ACPA status in early rheumatoid arthritis.

OBJECTIVES: To discover autoantibodies to non-modified proteins associated with the presence/absence of anticitrullinated protein antibodies (ACPA) in rheumatoid arthritis (RA). METHODS: The autoantibody repertoire of 80 ACPA negative and 80 ACPA positive RA subjects from the Swedish population-based Epidemiological Investigation of RA (EIRA) cohort was screened using a suspension bead array built on protein fragments earlier described as autoimmunity targets. Four autoantibodies positive in the initial screening were validated in another set of EIRA samples containing 317 ACPA-positive, 302 ACPA-negative and 372 age- and sex-matched controls. The relationship between the four autoantibodies and lung abnormalities on high-resolution computed tomography (HRTC) was examined in 93 early RA patients from LURA cohort. Association between the autoantibodies, smoking and MHC class II alleles was assessed by logistic regression analysis. RESULTS: : Anti-ANOS1 and anti-MURC IgG levels were associated with ACPA-positive status (OR = 3.02; 95% CI 1.87-4.89; and OR = 1.86; 95% CI 1.16-2.97, respectively) and increased in ACPA-positive patients compared with controls. Anti-ANOS1 IgG was associated with smoking habit (OR = 2.11; 95% CI 1.22-3.69) and anti-MURC IgG with the presence of the MHC class II "shared-epitope" genes (OR = 1.95; 95% CI 1.11-3.46). Anti-TSPYL4 IgG was associated with ACPA-negative (OR = 0.41; 95% CI 0.19-0.89). Anti-TSPYL4 IgG and anti-MAP2K6 IgG levels were increased in the ACPA-negative patients compared with controls. Presence of anti-MAP2K6 IgG and anti-TSPYL4 IgG correlated negatively with HRCT-defined lung abnormalities. CONCLUSIONS: These four autoantibodies may be useful in diagnostics and in predicting clinical phenotypes of RA.

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.

The association between autoantibodies and risk for venous thromboembolic events among patients with rheumatoid arthritis.

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.

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.

Distinct HLA Associations with Rheumatoid Arthritis Subsets Defined by Serological Subphenotype.

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.

Anti-citrullinated protein antibody specificities and pulmonary fibrosis in relation to genetic loci in early rheumatoid arthritis.

OBJECTIVES: Pulmonary manifestations in RA are common comorbidities, but the underlying mechanisms are largely unknown. The added value of a multiplex of ACPA and genetic risk markers was evaluated for the development of pulmonary fibrosis (PF) in an inception cohort. METHODS: A total of 1184 patients with early RA were consecutively included and followed prospectively from the index date until death or 31 December 2016. The presence of 21 ACPA fine specificities was analysed using a custom-made microarray chip (Thermo Fisher Scientific, Uppsala, Sweden). Three SNPs, previously found related to PF were evaluated, rs2609255 (FAM13A), rs111521887 (TOLLIP) and rs35705950 (MUC5B). ACPA and genetic data were available for 841 RA patients, of whom 50 developed radiologically defined PF. RESULTS: In unadjusted analyses, 11 ACPA specificities were associated with PF development. In multiple variable analyses, six ACPA specificities were associated with increased risk of PF: vimentin (Vim)60-75, fibrinogen (Fib)ß62-78 (72), Fibα621-635, Bla26, collagen (C)II359-369 and F4-CIT-R (P < 0.01 to P < 0.05). The number of ACPA specificities was also related to PF development (P < 0.05 crude and adjusted models). In multiple variable models respectively adjusted for each of the SNPs, the number of ACPA specificities (P < 0.05 in all models), anti-Vim60-75 (P < 0.05, in all models), anti-Fibß62-78 (72) (P < 0.001 to P < 0.05), anti-CII359-369 (P < 0.05 in all models) and anti-F4-CIT-R AQ4 (P < 0.01 to P < 0.05), anti-Fibα621-635 (P < 0.05 in one) and anti-Bla26 (P < 0.05 in two) were significantly associated with PF development. CONCLUSION: The development of PF in an inception cohort of RA patients was associated with both presence of certain ACPA and the number of ACPA specificities and risk genes.

Rheumatoid arthritis autoantibodies and their association with age and sex.

OBJECTIVES: To examine the association between individual rheumatoid arthritis (RA) autoantibodies, sex and age at RA onset. METHODS: Anti-CCP2, IgA-, IgG- and IgM-RF were analysed centrally in baseline sera from 1600 RA patients diagnosed within one year of RA symptom onset. Cut-offs for RF isotypes were determined at the 98th percentile based on RA-free controls, close to the 98.4% anti-CCP2 specificity. RESULTS: Anti-CCP2 was found in 1020 patients (64%), IgA RF in 692 (43%), IgG RF in 529 (33%) and IgM RF in 916 (57%) of the patients. When assessed one by one, anti-CCP2 and IgM RF were both associated with lower age at RA diagnosis. When assessed in one joint model, the association to IgM RF weakened and a strong association between IgA RF and higher age at RA diagnosis appeared. IgA RF and IgG RF associated with male sex, and IgM RF with female sex, with no difference for anti-CCP2. When the model was adjusted for sex, the association between IgM RF and age disappeared, whereas the strong associations between IgA RF and high age and between anti-CCP2 and low age at diagnosis remained. Further adjustments for smoking, shared epitope and inclusion year did not change the outcome. Univariate analyses stratified on anti-CCP2 and IgA RF status confirmed the findings. CONCLUSIONS: Anti-CCP associate with low, and IgA RF with high age at RA onset. RFs and anti-CCP2 display opposing association with sex. These results underscore that studies on RA phenotypes in relation to autoantibodies should accommodate age and sex.

Effects of B-cell directed therapy on the preclinical stage of rheumatoid arthritis: the PRAIRI study.

OBJECTIVES: We explored the effects of B-cell directed therapy in subjects at risk of developing autoantibodypositive rheumatoid arthritis (RA), who never experienced inflammatory arthritis before, and explored biomarkers predictive of arthritis development. METHODS: Individuals positive for both anti-citrullinated peptide antibodies and rheumatoid factor but without arthritis were included in a randomised, double-blind, placebo-controlled study to receive a single infusion of 1000 mg rituximab or placebo. RESULTS: Eighty-one individuals received treatment and were followed up for a mean of 29.0 (0-54) months, during which 30/81 (37%) individuals developed arthritis. The observed risk of developing arthritis in the placebo-treated group was 40%, which was decreased by 55% (HR 0.45, 95% CI 0.154 to 1.322) in the rituximab-treated group at 12 months. Rituximab treatment caused a delay in arthritis development of 12 months compared with placebo treatment at the point when 25% of the subjects had developed arthritis (p<0.0001). Erythrocyte sedimentation rate and the presence of anti-citrullinated α-enolase peptide 1 at baseline were significant predictors of arthritis development. CONCLUSIONS: A single infusion of 1000 mg rituximab significantly delays the development of arthritis in subjects at risk of developing RA, providing evidence for the pathogenetic role of B cells in the earliest, prearthritis stage of autoantibody positive RA.

Anticitrullinated protein/peptide antibody multiplexing defines an extended group of ACPA-positive rheumatoid arthritis patients with distinct genetic and environmental determinants.

INTRODUCTION: The 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. METHODS: We 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. RESULTS: The 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. CONCLUSIONS: Multiplexing 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.

Number of individual ACPA reactivities in synovial fluid immune complexes, but not serum anti-CCP2 levels, associate with inflammation and joint destruction in rheumatoid arthritis.

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.

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.

Multiplex analyses of antibodies against citrullinated peptides in individuals prior to development of rheumatoid arthritis.

OBJECTIVE: The presence of antibodies against cyclic citrullinated peptides has been demonstrated to precede the onset of symptoms of rheumatoid arthritis (RA) by several years. The aim of this study was to analyze antibodies against 10 citrullinated autoantigen-derived peptides for reactivity before the onset of RA symptoms. METHODS: A case-control study was conducted within the Medical Biobank of Northern Sweden. The study was performed in 409 individuals, 386 of whom donated 717 blood samples before the onset of symptoms of RA (pre-patients). The median period of time predating the onset of RA was 7.4 years. A total of 1,305 population-based control subjects were also studied. Antibodies to 10 citrullinated peptides, fibrinogen α573 (Fibα573), Fibα591, Fibß36-52, Fibß72, Fibß74, α-enolase (citrullinated α-enolase peptide 1 [CEP-1]), triple-helical type II collagen peptide C1 (citC1III), filaggrin, vimentin 2-17 (Vim2-17), and Vim60-75, were analyzed using a microarray system. RESULTS: The fluorescence intensity of antibodies against Fibß36-52, Fibß74, CEP-1, citC1III, and filaggrin was significantly increased in pre-patients compared with controls (P<0.001). The levels of the earliest-detectable antibodies (Fibα591 and Vim60-75) fluctuated over time, with only a slight increase after the onset of disease. The frequency of antibodies against Fibß36-52, CEP-1, and filaggrin increased gradually, reaching the highest levels before symptom onset. The frequency of a cluster of antibodies, citC1III, Fibα573, and Fibß74, increased only slightly before the onset of symptoms but increased prominently after disease onset. The odds ratio for the development of RA in individuals expressing both CEP-1 and Fibß36-52 antibodies (using data from samples obtained <3.35 years predating symptom onset) was 40.4 (95% confidence interval 19.8-82.3) compared with having either antibody alone. CONCLUSION: Development of an immune response toward citrullinated peptides is initially restricted but expands with time to induce a more specific response, with levels, particularly those of antibodies against CEP-1, Fibß36-52, and filaggrin, increasing during the predating time period closer to the onset of symptoms.

Gastrointestinal symptoms in patients with postural orthostatic tachycardia syndrome in relation to hemodynamic findings and immunological factors.

Gastrointestinal (GI) symptoms are common in postural orthostatic tachycardia syndrome (POTS). We aimed to explore the prevalence and severity of GI symptoms in POTS, and to investigate immunological factors, hemodynamic findings, and their possible association with GI symptoms in POTS. Forty-three patients (93% female, median age 30.6 (26.0-41.0) years), previously diagnosed with POTS and 74 healthy controls (78% female, median age 35.6 (28.8-41.7) years) were included. The participants completed a questionnaire including prevalence of GI symptoms, the irritable bowel syndrome severity scoring system (IBS-SSS), and visual analog scale for IBS (VAS-IBS). All POTS patients were previously examined by tilt test (2010-2021) and the vast majority with more recent active standing test (2017-2021), which included monitoring of heart rate (HR). ΔHR was calculated as difference between supine and upright position. Continuous variables from IBS-SSS and VAS-IBS were correlated to ΔHR. A microarray containing several autoantigens commonly targeted in systemic autoimmune disorders was used to assess prevalent autoantibodies in POTS and controls. Total IgE and S-tryptase were analyzed. GI symptoms were more prevalent and severe in POTS than in controls; nausea being the most prevalent (79.1% vs 4.9%, p < 0.001) and bloating and flatulence being the most severe (median 65 (25-88) vs 0 (0-14), p < 0.001). The median total IBS-SSS was 213 (135-319) in POTS vs 13 (0-54) in controls (p < 0.001). Total IBS-SSS was associated with low psychological wellbeing (r = 0.539, p < 0.001) in POTS. ΔHRmax correlated inversely with abdominal pain (r = -0.406, p = 0.007). After adjustments for psychological wellbeing, total IBS-SSS still associated inversely with ΔHR10min (ß: 4.748; 95% CI: -9.172 to -0.324; p = 0.036). Similar results were seen with active standing test. The prevalence of autoantibodies did not differ between POTS and controls (29.4% vs 33.3%, p = 0.803). There was no association between GI symptoms and autoantibody status. Total IgE and tryptase were elevated in a few cases. This study confirms the high prevalence of GI symptoms in POTS. More pronounced tachycardia upon tilt table testing seems to be inversely correlated with severity of chronic GI symptoms in POTS. This study did not support the hypothesis that POTS is associated with immunological factors.

The impact of immunomodulating treatment on the immunogenicity of COVID-19 vaccines in patients with immune-mediated inflammatory rheumatic diseases compared to healthy controls. A Swedish nationwide study (COVID19-REUMA).

OBJECTIVES: To elucidate antibody responses after the second and third dose of COVID-19 vaccine in patients with inflammatory rheumatic diseases (IRD) treated with biologic/targeted disease modifying anti-rheumatic drugs (b/ts DMARDs). METHODS: Antibody levels to antigens representing spike full length protein and spike S1 were measured before vaccination, 2-12 weeks after the second dose, before and after the third dose using multiplex bead-based serology assay. Positive antibody response was defined as antibody levels over cut off (seropositivity) in seronegative individuals or ≥ 4-fold increase in antibodies in individuals seropositive for both spike proteins. RESULTS: Patients (n = 414) receiving b/ts DMARDs (283 had arthritis, 75 systemic vasculitis and 56 other autoimmune diseases) and controls (n = 61) from five Swedish regions participated. Treatments groups were: rituximab (n = 145); abatacept (n = 22); Interleukin 6 receptor inhibitors [IL6i (n = 79)]; JAnus Kinase Inhibitors [JAKi (n = 58)], Tumour Necrosis Factor inhibitor [TNFi (n = 68)] and Interleukin12/23/17 inhibitors [IL12/23/17i (n = 42)]. Percentage of patients with positive antibody response after two doses was significantly lower in rituximab (33,8%) and abatacept (40,9%) (p < 0,001) but not in IL12/23/17i, TNFi or JAKi groups compared to controls (80,3%). Higher age, rituximab treatment and shorter time between last rituximab course and vaccination predicted impaired antibody response. Antibody levels collected 21-40 weeks after second dose decreased significantly (IL6i: p = 0,02; other groups: p < 0,001) compared to levels at 2-12 week but most participants remained seropositive. Proportion of patients with positive antibody response increased after third dose but was still significantly lower in rituximab (p < 0,001). CONCLUSIONS: Older individuals and patients on maintenance rituximab have an impaired response after two doses of COVID-19 vaccine which improves if the time between last rituximab course and vaccination extends and also after an additional vaccine dose. Rituximab patients should be prioritized for booster vaccine doses. TNFi, JAKi and IL12/23/17i does not diminished humoral response to primary and an additional vaccination.

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.

Anti-Citrullinated Protein Antibody Reactivity towards Neutrophil-Derived Antigens: Clonal Diversity and Inter-Individual Variation.

BACKGROUND: Why the adaptive immune system turns against citrullinated antigens in rheumatoid arthritis (RA) and whether anti-citrullinated protein antibodies (ACPAs) contribute to pathogenesis are questions that have triggered intense research, but still are not fully answered. Neutrophils may be crucial in this context, both as sources of citrullinated antigens and also as targets of ACPAs. To better understand how ACPAs and neutrophils contribute to RA, we studied the reactivity of a broad spectrum of RA patient-derived ACPA clones to activated or resting neutrophils, and we also compared neutrophil binding using polyclonal ACPAs from different patients. METHODS: Neutrophils were activated by Ca2+ ionophore, PMA, nigericin, zymosan or IL-8, and ACPA binding was studied using flow cytometry and confocal microscopy. The roles of PAD2 and PAD4 were studied using PAD-deficient mice or the PAD4 inhibitor BMS-P5. RESULTS: ACPAs broadly targeted NET-like structures, but did not bind to intact cells or influence NETosis. We observed high clonal diversity in ACPA binding to neutrophil-derived antigens. PAD2 was dispensable, but most ACPA clones required PAD4 for neutrophil binding. Using ACPA preparations from different patients, we observed high patient-to-patient variability in targeting neutrophil-derived antigens and similarly in another cellular effect of ACPAs, the stimulation of osteoclast differentiation. CONCLUSIONS: Neutrophils can be important sources of citrullinated antigens under conditions that lead to PAD4 activation, NETosis and the extrusion of intracellular material. A substantial clonal diversity in targeting neutrophils and a high variability among individuals in neutrophil binding and osteoclast stimulation suggest that ACPAs may influence RA-related symptoms with high patient-to-patient variability.