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Objectives: To investigate the occurrence and dynamics of secretory component-containing antibodies towards citrullinated proteins (SC ACPA) in plasma from pre-symptomatic individuals subsequently developing rheumatoid arthritis (RA).

Methods: We studied 319 individuals who had donated plasma prior to RA onset (median predating time 4.7 years), whereof 181 also donated samples after diagnosis. One hundred individuals were randomly selected from the same biobank cohorts to serve as controls. SC ACPA, total secretory IgA (TSIgA) and IgG ACPA were analysed in plasma by enzyme-linked immunoassays.

Results: Circulating SC ACPA levels in pre-symptomatic individuals and RA patients were significantly increased compared with controls [median (interquartile range) 108 (108), 179 (248) and 12.5 (537) AU/ml, respectively; P < 0.001], and SC ACPA levels in RA patients were significantly increased compared with pre-symptomatic individuals (P < 0.001). SC ACPA increased, in terms of both levels and proportion of positive samples, closer to symptom onset and diagnosis. TSIgA was not elevated compared with controls either during the pre-dating time or after diagnosis. The earliest detected SC ACPA positive sample was 9 years before symptom onset, as compared with 11 years for IgG ACPA. Only two pre-dating samples were positive for SC ACPA and negative for IgG ACPA.

Conclusions: Circulating SC ACPA responses arise and magnify during the asymptomatic phase of disease development in a subgroup of RA patients. This suggests mucosal involvement prior to both symptom onset and subsequent arthritis. As mirrored in the circulation, however, SC ACPA does not seem to precede the IgG ACPA response.

Objective: Platelet activation is thought to participate in polymyalgia rheumatica (PMR) pathogenesis. Upon platelet activation, mitochondria are expelled into the extracellular space. However, whether extracellular mitochondria are present in patients with PMR and whether they can induce platelet activation is not known.

Methods: To investigate this, we measured markers of platelet activation (thrombospondin-1 [TSP-1]), mitochondrial-derived N-formyl methionine peptide (fMET), and autoantibodies directed toward specific mitochondrial antigen mitofusin-1 (MFN1) by enzyme-linked immunosorbent assay in plasma of healthy controls (HCs, n = 30) and patients with PMR without giant cell arteritis (GCA) (n = 45) and patients with PMR with GCA (n = 9) before and after treatment with glucocorticoid therapy. Ultrapure mitochondria were opsonized with plasma from patients with PMR without GCA (n = 45) or HCs (n = 10) and were subsequently incubated with HC platelets. Platelet activation was assessed by P-selectin levels using flow cytometry.

Results: Plasma levels of anti-MFN1 IgG were elevated in patients with PMR with and without GCA before glucocorticoid therapy when compared with HCs (P < 0.01 for both groups). Levels of anti-MFN1 IgG significantly reduced after treatment with glucocorticoids in both groups (P < 0.01). Levels of fMET were also significantly higher in patients with PMR with and without GCA before glucocorticoid therapy in comparison with HCs (P < 0.001 and P < 0.01, respectively). However, the levels of fMET only dropped significantly after therapy in patients with PMR without GCA (P < 0.001). Plasma levels of TSP-1 were elevated in patients with PMR with and without GCA before glucocorticoid therapy when compared to HC (P < 0.001 for both groups). After glucocorticoid therapy, plasma levels of TSP-1 decreased significantly only in patients with PMR without GCA (P = 0.023). Mitochondria opsonized with plasma from patients with PMR without GCA induced higher platelet activation regardless of treatment status as compared with plasma from HCs (P < 0.0001 and P < 0.01 for pretreatment and posttreatment).

Conclusion: Our results indicate increased platelet activation and the presence of mitochondrial antigens and antibodies in the circulation of patients with PMR. Blocking mitochondrial-mediated platelet activation may reduce inflammation in patients with PMR, with potential therapeutic implications.

OBJECTIVE: Neutrophils are important in host defence. However, neutrophils are also linked to inflammation and organ damage. The purpose of this study was to assess whether markers of neutrophil activation are increased in PMR.

METHODS: Levels of immune complexes (IC), calprotectin and neutrophil extracellular traps (NETs) were measured in plasma of healthy individuals (n = 30) and patients with PMR (n = 60), at flare and upon treatment with glucocorticoids using ELISA. Plasma-mediated neutrophil activation was assessed in presence of an FcγRIIA inhibitory antibody (IV.3).

RESULTS: Plasma levels of calprotectin and NETs were elevated in PMR (P < 0.001). Mechanistically, neutrophil activation was driven by ICs, present in plasma, able to up-regulate neutrophil activation markers CD66b and CD11b (P < 0.0001) in an FcγRIIA-dependent manner (P < 0.01). Of note, circulating levels of IC correlated with plasma induced CD66b and CD11b (r = 0.51, P = 0.004, and r = 0.46, P = 0.01, respectively) and decreased after glucocorticoid therapy. In contrast to NETs, calprotectin significantly decreased after glucocorticoid therapy (P < 0.001) and was higher in PMR without overlapping GCA compared with patients with overlapping disease (P = 0.014). Interestingly, musculoskeletal involvement was associated with elevated levels of calprotectin before initiation of glucocorticoid therapy (P = 0.036).

CONCLUSIONS: Neutrophil activation, including NET formation, is increased in PMR, through IC-mediated engagement of FcγRIIA. Clinically, neutrophil activation is associated with musculoskeletal involvement, with calprotectin, but not NETs, being a biomarker of treatment response in PMR patients. In all, IC-mediated neutrophil activation is a central process in PMR pathogenesis identifying potential novel therapeutic targets (FcγRIIA), as well as soluble markers for disease monitoring (calprotectin).

Objectives: To identify the arthritogenic B cell epitopes of glucose-6-phosphate isomerase (GPI) and their association with rheumatoid arthritis (RA).

Methods: IgG response towards a library of GPI peptides in patients with early RA, pre-symptomatic individuals and population controls, as well as in mice, were tested by bead-based multiplex immunoassays and ELISA. Monoclonal IgG were generated, and the binding specificity and affinity were determined by ELISA, gel size exclusion chromatography, surface plasma resonance and X-ray crystallography. Arthritogenicity was investigated by passive transfer experiments. Antigen-specific B cells were identified by peptide tetramer staining.

Results: Peptide GPI293-307 was the dominant B cell epitope in K/BxN and GPI-immunised mice. We could detect B cells and low levels of IgM antibodies binding the GPI293-307 epitopes, and high affinity anti-GPI293-307 IgG antibodies already 7 days after GPI immunisation, immediately before arthritis onset. Transfer of anti-GPI293-307 IgG antibodies induced arthritis in mice. Moreover, anti-GPI293-307 IgG antibodies were more frequent in individuals prior to RA onset (19%) than in controls (7.5%). GPI293-307-specific antibodies were associated with radiographic joint damage. Crystal structures of the Fab-peptide complex revealed that this epitope is not exposed in native GPI but requires conformational change of the protein in inflamed joint for effective recognition by anti-GPI293-307 antibodies.

Conclusions: We have identified the major pathogenic B cell epitope of the RA-associated autoantigen GPI, at position 293-307, exposed only on structurally modified GPI on the cartilage surface. B cells to this neo-epitope escape tolerance and could potentially play a role in the pathogenesis of RA.

Background: The increased comorbidity and mortality in rheumatoid arthritis (RA) patients are largely due to cardiovascular disease (CVD). Previously, we demonstrated increased frequencies of risk factors for CVD (elevated body mass index (BMI), elevated apoliprotein (Apo) B:ApoA1 ratio, and smoking) in pre-RA individuals compared with matched controls. Objectives: Assess the impact of traditional CV risk factors present before the onset of RA on the risk of CV events (CVE) after diagnosis in comparison with matched controls. Methods: A case–control study including 521 pre-symptomatic individuals and 1566 controls identified within the Health Surveys of the Medical Biobank was performed. CVD risk factors were hypertension, elevated ApoB:A1 ratio, BMI, diabetes, and smoking. Information on comorbidities was requested from the Swedish National Patient Register and Cause of Death Register. Results: Pre-RA individuals had a higher risk of future CVE compared with matched controls (HR [95% CI] 1.70 [1.31–2.21]), which remained after adjustments for risk factors for CVD (HR [95% CI] 1.73 [1.27–2.35]). Most risk factors were associated with CVE after diagnosis, and a combination resulted in a higher risk in RA compared with controls; two risk factors, HR [95% CI] 2.70 [1.19–6.13] vs. 1.26 [0.75–2.13]; and three to four risk factors, HR [95% CI] 6.32 [2.92–13.68] vs. 3.77 [2.34–6.00]. Conclusions: Risk factors for CVD present in pre-RA individuals were associated with future CVE, and even after adjustments for these risk factors and treatments after RA onset, pre-RA individuals had a higher risk of CVE compared with controls. These findings further highlight the importance of the early assessment of risk for CVD.

Objective: Increased soluble levels of complement effectors have been demonstrated in active anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), but the timing of complement activation in the autoimmune inflammation remains elusive. This study investigated whether the complement system is activated before onset of symptoms in AAV.

Method: The Swedish National Patient Register and Cause of Death register were linked to registers of five biobanks to identify individuals sampled before AAV symptom onset. Diagnosis of AAV and time-point for symptom onset were confirmed by reviewing medical records. We identified 64 presymptomatic individuals with serum samples > 1 month < 10 years from AAV symptom onset and 122 matched controls. Complement factors (C2, C5) and activation markers (C5a, C4b) were measured using Luminex technology.

Results: Presymptomatic individuals had higher levels of C5 up to 6.5 years before symptom onset, compared with controls [median (IQR) 80.7 (131.9) vs 46.6 (63.4) µg/mL, p = 0.05]. Levels of C5a increased significantly during the pre-dating time (p = 0.033) until symptom onset. The complement levels were significantly higher in presymptomatic myeloperoxidase (MPO)-ANCA⁺ individuals versus MPO-ANCA⁻ and proteinase-3-ANCA⁺ individuals. C5 was significantly increased in cases with renal involvement at diagnosis versus controls (p = 0.022), whereas levels of both C5 and C5a were significantly increased in presymptomatic individuals diagnosed with microscopic polyangiitis after onset compared with controls (C5: p = 0.027; C5a: p = 0.027).

Conclusion: Activation of the complement system is an early event in the pathogenesis of AAV and is mainly associated with MPO-ANCA⁺ AAV and with microscopic polyangiitis.

Microbiota has been associated with autoimmune diseases, with nasal Staphylococcus aureus being implicated in the pathogenesis of anti-neutrophil cytoplasmic antibody–associated vasculitis (AAV). Little is known about the role of oral microbiota in AAV. In this study, levels of IgG antibodies to 53 oral bacterial species/subspecies were screened using immunoblotting in plasma/serum in pre-symptomatic AAV-individuals (n = 85), matched controls, and established AAV-patients (n = 78). Saliva microbiota from acute-AAV and controls was sequenced from 16s rDNA amplicons. Information on dental status was extracted from a national register. IgG levels against oral bacteria were lower in established AAV versus pre-AAV and controls. Specifically, pre-AAV samples had, compared to controls, a higher abundance of periodontitis-associated species paralleling more signs of periodontitis in established AAV-patients than controls. Saliva microbiota in acute-AAV showed higher within-sample diversity but fewer detectable amplicon-sequence variants and taxa in their core microbiota than controls. Acute-AAV was not associated with increased abundance of periodontal bacteria but species in, e.g., Arthrospira, Staphylococcus, Lactobacillus, and Scardovia. In conclusion, the IgG profiles against oral bacteria differed between pre-AAV, established AAV, and controls, and microbiota profiles between acute AAV and controls. The IgG shift from a pre-symptomatic stage to established disease cooccurred with treatment of immunosuppression and/or antibiotics.

We recently reported that increased serum adiponectin was associated with rheumatoid arthritis (RA) risk in subjects with obesity. We hereby aim to determine if other adipokines associate with RA risk and if the association between adiponectin and RA is independent of other adipokines. Two nested-case control studies were performed in two different cohorts: 82 participants of the Swedish Obese Subjects (SOS) study who developed RA during follow-up matched with 410 controls, and 88 matched pairs from the Medical Biobank of Northern Sweden. Baseline levels of circulating adipokines were measured using ELISA. In a multivariable analysis in the SOS cohort, higher adiponectin was associated with an increased risk of RA independently of other adipokines (OR for RA risk: 1.06, 95% CI: 1.01-1.12, p = 0.02). No association between leptin, resistin, and visfatin levels and the risk of RA was detected. In the cohort from the Medical Biobank of Northern Sweden, higher adiponectin was associated with an increased risk of RA only in participants with overweight/obesity (OR: 1.17, 95% CI: 1.01-1.36, p = 0.03), independently of other adipokines. Our results show that in individuals with overweight/obesity, higher circulating levels of adiponectin, but not leptin, resistin, or visfatin, were associated with an increased RA risk.

Objectives: Anti-neutrophil cytoplasmic autoantibodies [ANCA) are important for diagnosis of ANCA-associated vasculitides (AAV). The timing of antibody development is not well established. To investigate the development of proteinase 3 (PR3)- and myeloperoxidase (MPO)-ANCA, blood samples collected before onset of symptoms of AAV were analysed.

Methods: To identify AAV patients with blood samples predating symptoms, the National Patient Register and Cause of Death register were scrutinized for ICD codes for AAV and linked to the registers of five biobanks. Diagnoses of AAV and time point for symptom onset were confirmed by reviewing 504 case-record. Eighty-five AAV cases (34 males, 51 females) with samples >1 month < 10 years from AAV symptom onset and two controls matched for sex, age, and sampling time for each case were included. Samples were screened using ELISAs for ANCA and further analysed for PR3-or MPO- specificities.

Results: In ANCA-screen 35.7% of the pre-symptomatic cases and 3.5% of controls tested positive (p < 0.01). 26.2% of the cases were PR3-ANCA+ and 10.7% MPO-ANCA+. Median (Q1-Q3) predating time for PR3-ANCA+ was 2.7 (0.3–7.7) years and MPO-ANCA+ 2.0 (0.9–3.5) years. PR3-ANCA was demonstrated in samples up to nine years before symptom onset. At symptom onset predating PR3-ANCA+ cases were younger than PR3-ANCA- (P < 0.01), and MPO-ANCA+ were older than MPO-ANCA- (p < 0.05). Predating MPO-ANCA+ cases vs. MPO-ANCA- and vs. PR3-ANCA+ cases had more often at symptoms onset manifestations from lungs, kidneys or peripheral nervous system (p < 0.01 and p < 0.05, respectively).

Conclusion: The PR3-and MPO-ANCAs are present years before AAV symptom onset and represent distinct diseases.

Objective: Psoriatic arthritis (PsA) is a heterogeneous inflammatory disease associated with psoriasis. Underlying genetic factors are considered important for disease expression and prognosis of PsA. Interleukin-1 beta-regulating protein complexes called inflammasomes are associated with several inflammatory diseases, e.g. rheumatoid arthritis and psoriasis. The aim was to determine whether inflammasome-related genetic variation is associated with PsA susceptibility or different disease phenotypes.

Method: DNA from 724 patients with PsA and 587 population-based controls from northern Sweden was analysed for single-nucleotide polymorphisms in NLRP3-Q750K (rs35829419), NLRP3 (rs10733113), CARD8-C10X (rs2043211), NLRP1 (rs8079034), and NLRP1 (rs878329).

Results: Significant associations were found with the genotype AA (vs AT+TT) of rs2043211 for PsA patients compared with controls [odds ratio (OR), 95% confidence interval (CI) 1.32 (1.05-1.65), p = 0.016]; and between the C-allele of rs878329 and axial involvement of PsA [OR (95% CI) 1.37 (1.02-1.84), p = 0.035], the T-allele of rs8079034 with prescription of conventional synthetic disease-modifying anti-rheumatic drugs [OR (95% CI) 1.76 (1.23-2.53), p = 0.0020], the G-allele of rs10733113 and patients with a skin disease with early onset [OR (95% CI) 1.58 (1.13-2.21), p = 0.007], and the C-allele of rs35829419 and a destructive/deforming disease [OR (95% CI) 1.63 (1.04-2.55), p = 0.030].

Conclusions: This study is the first to show an association with a genetic polymorphism in an inflammasome-related gene, CARD8-C10X (rs2043211), in patients with PsA. Associations between different phenotypes of PsA and different polymorphisms of the inflammasome genes were also found. Our results indicate the involvement of inflammasome genes in the pathogenesis and disease expression of PsA.