IntroductionSmoking is a well-established risk factor for rheumatoid arthritis (RA), and it has been proposed that smoking-induced citrullination renders autoantigens immunogenic. To investigate this mechanism, we examined human lung tissue from 40 subjects with defined smoking status, with or without chronic obstructive pulmonary disease (COPD), and control tissues from other organs for citrullinated proteins and the deiminating enzymes peptidylarginine deiminase type-2 (PAD2) and -4 (PAD4).MethodsLung tissue samples, dissected from lobectomy specimens from 10 never smokers, 10 smokers without airflow limitation, 13 COPD smokers and eight COPD ex-smokers, and control tissue samples (spleen, skeletal muscle, liver, ovary, lymph node, kidney and heart), were analysed for citrullinated proteins, PAD2 and PAD4 by immunoblotting. Citrulline and homocitrulline residues in enolase and vimentin were analysed by partial purification by gel electrophoresis followed by mass spectrometry in 12 of the lung samples and one from each control tissues. Band intensities were scored semi-quantitatively and analysed by two-tailed Mann-Whitney T-test.ResultsWithin the lung tissue samples, citrullinated proteins, PAD2 and PAD4 were found in all samples, with an increase in citrullination in COPD (P = 0.039), but minimal difference between smokers and non-smokers (P = 0.77). Citrullination was also detected at lower levels in the tissues from other organs, principally in lymph node, kidney and skeletal muscle. Mass spectrometry of the lung samples showed that vimentin was citrullinated at positions 71, 304, 346, 410 and 450 in non-smokers and smokers both with and without COPD. A homocitrulline at position 104 was found in four out of six COPD samples and one out of six non-COPD. Citrulline-450 was also found in three of the control tissues. There were no citrulline or homocitrulline residues demonstrated in α-enolase.ConclusionsWe have shown evidence of citrullination of vimentin, a major autoantigen in RA, in both non-smokers and smokers. The increase in citrullinated proteins in COPD suggests that citrullination in the lungs of smokers is mainly due to inflammation. The ubiquity of citrullination of vimentin in the lungs and other tissues suggests that the relationship between smoking and autoimmunity in RA may be more complex than previously thought.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0520-x) contains supplementary material, which is available to authorized users.

BackgroundPeriodontitis (PD) is a known risk factor for rheumatoid arthritis (RA) and there is increasing evidence that the link between the two diseases is due to citrullination by the unique bacterial peptidylarginine deiminase (PAD) enzyme expressed by periodontal pathogen Pophyromonas gingivalis (PPAD). However, the precise mechanism by which PPAD could generate potentially immunogenic peptides has remained controversial due to lack of information about the structural and catalytic mechanisms of the enzyme.ObjectivesBy solving the 3D structure of PPAD we aim to characterise activity and elucidate potential mechanisms involved in breach of tolerance to citrullinated proteins in RA.MethodsPPAD and a catalytically inactive mutant PPADC351A were crystallised and their 3D structures solved. Key residues identified from 3D structures were examined by mutations. Fibrinogen and α-enolase were incubated with PPAD and P. gingivalis arginine gingipain (RgpB) and citrullinated peptides formed were sequenced and quantified by mass spectrometry.ResultsHere, we solve the crystal structure of a truncated, highly active form of PPAD. We confirm catalysis is mediated by the following residues: Asp130, His236, Asp238, Asn297 and Cys351 and show Arg152 and Arg154 may determine the substrate specificity of PPAD for C-terminal arginines. We demonstrate the formation of 37 C-terminally citrullinated peptides from fibrinogen and 11 from α-enolase following incubation with tPPAD and RgpB.ConclusionsPPAD displays an unequivocal specificity for C-terminal arginine residues and readily citrullinates peptides from key RA autoantigens. The formation of these novel citrullinated peptides may be involved in breach of tolerance to citrullinated proteins in RA.

Objective In addition to the long-established link with smoking, periodontitis (PD) is also a risk factor for rheumatoid arthritis (RA). To elucidate the mechanism by which PD could induce antibodies to citrullinated peptides (ACPA), we examine the antibody response to a novel citrullinated peptide from cytokeratin type I 13 identified in gingival crevicular fluid (GCF), and compare the response to 4 other citrullinated peptides in patients with RA, well-characterized for PD and smoking. Methods The citrullinomes of GCF and periodontal tissue from people with PD were mapped by mass spectrometry. Antibodies to citrullinated peptides from cytokeratin type I 13 (cCK13), tenascin-C (cTNC5), vimentin (cVIM), enolase (CEP-1) and fibrinogen β (cFIBβ) were examined by ELISA in patients with RA (n=287) and osteoarthritis (OA) (n=330), and cross-reactivity assessed by inhibition assays. Results A novel citrullinated peptide cCK13-1 (444TSNASGR-cit-TSDV-cit-RP458) identified in GCF, exhibited elevated antibody responses in RA patients (24%). Anti-cCK13-1 antibodies correlated with anti-cTNC5 antibodies, and absorption experiments confirmed this was not due to cross-reactivity. Only anti-cCK13-1 and anti-cTNC5 were associated with antibodies to the periodontal pathogen Prevotella intermedia (p=0.05 and p =0.001 respectively), but not with antibodies to Porphyromonas gingivalis arginine gingipains. Antibodies to CEP-1, cFIBβ and cVIM correlated with each other, and with smoking and shared epitope risk factors in RA. Conclusion This study identifies two groups of ACPA fine specificities associated with different RA risk factors; one predominantly linked to smoking and shared epitope, the other linking anti- cTNC5 and cCK13-1 to infection with the periodontal pathogen P. intermedia.

Performance of synovial tissue biopsies by rheumatologists in the US is feasible and generates high-quality samples for research. Through the use of cutting-edge technologies to analyze synovial biopsy specimens in conjunction with corresponding clinical information, a precision medicine-based approach for patients with RA is attainable.