The periodontal pathogen Porphyromonas gingivalis is hypothesized to be important in rheumatoid arthritis (RA) aetiology by inducing production of anti-citrullinated protein antibodies (ACPA). We have shown that ACPA precede RA onset by years, and that anti-P. gingivalis antibody levels are elevated in RA patients. The aim of this study was to investigate whether anti-P. gingivalis antibodies pre-date symptom onset and ACPA production. A case–control study (251 cases, 198 controls) was performed within the Biobank of Northern Sweden. Cases had donated blood samples (n = 422) before the onset of RA symptoms by 5.2 (6.2) years (median (interquartile range)). Blood was also collected from 192 RA patients following diagnosis. Antibodies against P. gingivalis virulence factor arginine gingipainB (RgpB), and a citrullinated peptide (CPP3) derived from the P. gingivalis peptidylarginine deiminase enzyme, were analysed by ELISA. Anti-RgpB IgG levels were significantly increased in pre-symptomatic individuals (mean ± SEM; 152.7 ± 14.8 AU/ml) and in RA patients (114.4 ± 16.9 AU/ml), compared with controls (p < 0.001). Anti-CPP3 antibodies were detected in 5 % of pre-symptomatic individuals and in 8 % of RA patients, with elevated levels in both subsets (4.33 ± 0.59 and 9.29 ± 1.81 AU/ml, respectively) compared with controls (p < 0.001). Anti-CPP3 antibodies followed the ACPA response, with increasing concentrations over time, whilst anti-RgpB antibodies were elevated and stable in the pre-symptomatic individuals with a trend towards lower levels after RA diagnosis. Anti-P. gingivalis antibody concentrations were significantly increased in RA patients compared with controls, and were detectable years before onset of symptoms of RA, supporting an aetiological role for P. gingivalis in the development of RA.

Rheumatoid arthritis (RA) is the most common autoimmune inflammatory disease, and single periodontitis-associated bacteria have been suggested in disease manifestation. Here, the oral microbiota was characterized in relation to the early onset of RA (eRA) taking periodontal status into consideration. 16S rRNA gene amplicon sequencing of saliva bacterial DNA from 61 eRA patients without disease-modifying anti-rheumatic drugs and 59 matched controls was performed. Taxonomic classification at 98.5% was conducted against the Human Oral Microbiome Database, microbiota functions were predicted using PICRUSt, and periodontal status linked from the Swedish quality register for clinically assessed caries and periodontitis. The participants were classified into three distinct microbiota-based cluster groups with cluster allocation differences by eRA status. Independently of periodontal status, eRA patients had enriched levels of Prevotella pleuritidis, Treponema denticola, Porphyromonas endodontalis and Filifactor alocis species and in the Porphyromonas and Fusobacterium genera and functions linked to ornithine metabolism, glucosylceramidase, beta-lactamase resistance, biphenyl degradation, fatty acid metabolism and 17-beta-estradiol-17-dehydrogenase metabolism. The results support a deviating oral microbiota composition already in eRA patients compared with healthy controls and highlight a panel of oral bacteria that may be useful in eRA risk assessment in both periodontally healthy and diseased persons.

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

Periodontal disease has been implicated in the pathogenesis of rheumatoid arthritis (RA), an autoimmune disease characterized by immune-mediated synovial damage, and antibodies to citrullinated antigens. Here, we investigate the association between exposure to the periodontal pathogen Aggregatibacter actinomycetemcomitans (Aa) and the development of RA. IgM, IgG and IgA antibodies to Aa leukotoxin A (LtxA) were detected by ELISA in plasma from a cohort of Swedish adults at different stages of RA development, from before onset of symptoms to established disease. Patients with early and established RA had increased levels of anti-LtxA IgM compared with matched non-RA controls and periodontally healthy individuals. Logistic regression revealed that anti-LtxA IgM levels were associated with RA during early disease (OR 1.012, 95%CI 1.007, 1.017), which was maintained after adjustment for smoking, anti-CCP antibodies, rheumatoid factor, HLA-DRB1 shared epitope alleles and sex. We found no association between anti-LtxA IgG/IgA antibodies and RA at any stage of disease development. The data support a temporal association between anti-LtxA IgM antibodies and the development of RA, suggesting that a subset of RA patients may have been exposed to Aa around the time of transition from being asymptomatic to become a patient with RA.

Vitamin D has been implicated as being involved in the aetio-pathogenesis of several autoimmune diseases including rheumatoid arthritis (RA). Previous studies present contradictory results. Vitamin D binding protein (DBP), the major transport protein, is also involved in various inflammatory processes. The aim of this study was to investigate the relationship between circulating levels of 25-hydroxyvitamin D [25(OH) D], DBP and polymorphisms in group-specific component (GC) in pre-symptomatic individuals and matched controls within prospective cohorts of the Northern Sweden. Blood samples donated to the Medical Biobank prior to the onset of symptoms of RA (n = 515, mean [SD] time before the onset of symptoms 6.2 [9.3] years) and from matched (2:1) population-based controls (n = 267) were used. Plasma 25(OH) vitamin D levels were analyzed using liquid chromatography tandem-mass spectrometry and DBP levels were analyzed using enzyme-linked immunosorbent assay. GC polymorphisms (rs4588 and rs7041) were analyzed with TaqMan assays (Applied Biosystems). Levels of 25(OH) D or DBP were not statistically different between pre-symptomatic individuals and controls in a crude, or a multiple-adjusted logistic regression model. However, an increased risk for future RA was found in females of DBP (OR 1.014 [95%CI 1.001–1.028]) per 10 mg/L adjusted for carriage of the minor allele of rs4588, in a multiple-adjusted model (p < 0.05). This study indicated that vitamin D is not associated with the future risk of RA although increasing levels of DBP were however, associated with an increased risk of disease in females carrying the minor allele of a DBP encoding SNP.

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 GPI_293-307 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 GPI_293-307 epitopes, and high affinity anti-GPI_293-307 IgG antibodies already 7 days after GPI immunisation, immediately before arthritis onset. Transfer of anti-GPI_293-307 IgG antibodies induced arthritis in mice. Moreover, anti-GPI_293-307 IgG antibodies were more frequent in individuals prior to RA onset (19%) than in controls (7.5%). GPI_293-307-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-GPI_293-307 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.