Anna B Montgomery

Expression of citrulline and homocitrulline residues in the lungs of non-smokers and smokers: implications for autoimmunity in rheumatoid arthritis.

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.

Crystal structure of Porphyromonas gingivalis peptidylarginine deiminase: implications for autoimmunity in rheumatoid arthritis

Background Periodontitis (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. Objectives By 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. Methods PPAD 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. Results Here, 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. Conclusions PPAD 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.

The case for measuring antibodies to specific citrullinated antigens.

Anti-citrullinated protein/peptide antibodies (ACPA) are the principal autoantibody system associated with rheumatoid arthritis (RA), with diagnostic sensitivity of 70% and specificity of 95%. Current testing for ACPA uses the anti-cyclic citrullinated peptide assay (anti-CCP) which measures a generalized reactivity with citrulline-containing peptides, thus giving no insight into reactivity to specific RA antigens. Of these, the best characterized are, α-enolase, fibrinogen/fibrin, vimentin, Type 2 collagen and filaggrin, antibodies to each of which are found in approximately 30–60% of RA cases. Given reports of cross-reactivity between citrullinated antigens, we discuss whether or not measuring these specific antibodies could aid: clinical diagnosis, identification of clinical subsets and drug responses, or provide insight into pathogenic mechanisms or etiology of RA.

Is Citrullination the Missing Link between Periodontal Disease and Rheumatoid Arthritis

Connections between periodontitis (PD) and rheumatoid arthritis (RA) are suggested by an increased prevalence of PD in RA, shared environmental and genetic risk factors, and correlating levels of severity when the two diseases occur together. Here, we compare and contrast the results from numerous studies documenting this association, and highlight the involvement of citrullination in the development of autoimmunity leading to the destructive pathology that characterizes RA. The contribution of citrullination to RA may occur in at least three distinct phases: (i) the initial breakdown of tolerance leading to autoimmunity; (ii) the maturation of the citrulline specificity of the autoantibody response; and (iii) the pro-inflammatory effect of citrullinated proteins themselves in established disease. We conclude that citrullination is more than a ‘missing link’; rather, it is an active process in the evolution of low levels of autoimmunity found in PD into the pathogenic anti-citrullinated protein response specific to RA.

Porphyromonas gingivalis Peptidyl Arginine Deiminase: A Unique Bacterial PAD with Implications for Periodontal Disease and Rheumatoid Arthritis

Recently, peptidyl arginine deiminase from Porphyromonas gingivalis (PPAD) has become a subject of an intense research. Despite catalyzing the reaction of citrullination (deimination) and being a member of the guanidino-group modifying superfamily of enzymes, PPAD and the mammalian peptidyl arginine deiminases (PAD/PADI) are phylogenetically unrelated proteins. The citrullination of host and bacterial proteins by PPAD has been implicated in the pathogenesis of periodontitis (PD) and postulated as the link between PD and rheumatoid arthritis (RA). In this chapter, we discuss the role of protein citrullination by PPAD in the pathogenesis of PD and RA.

Association of Distinct Fine Specificities of Anti-Citrullinated Peptide Antibodies With Elevated Immune Responses to Prevotella intermedia in a Subgroup of Patients With Rheumatoid Arthritis and Periodontitis.

In addition to the long‐established link with smoking, periodontitis (PD) is a risk factor for rheumatoid arthritis (RA). This study was undertaken to elucidate the mechanism by which PD could induce antibodies to citrullinated peptides (ACPAs), by examining the antibody response to a novel citrullinated peptide of cytokeratin 13 (CK‐13) identified in gingival crevicular fluid (GCF), and comparing the response to 4 other citrullinated peptides in patients with RA who were well‐characterized for PD and smoking.

07.14 Novel polymorphism of peptidylarginine deiminase from p. gingivalis augments bacterial pathogenicity and severity of periodontitis

Background Periodontitis (PD) is the most common chronic inflammatory disease caused by bacterial infection resulting in alveolar bone resorption and tooth loss.1,2 A main causative agent of PD is Porphyromonas gingivalis (Pg). 3,4 This periodontopathogen produces peptidylarginine deiminase (PPAD) catalysing conversion of arginine to citrulline.5,6 PD shares common mechanism and risk factors with rheumatoid arthritis (RA). Due to the presence of pathogenic autoantibodies reactive with citrullinated proteins in RA, citrullination of host and bacterial proteins by PPAD was proposed as a mechanistic link between PD and RA.7–9 The aim of this study was to investigate the impact of a novel polymorphism identified in the PPAD gene on bacterial virulence and PD clinical status. Materials and methods Gingival crevicular fluid (GCF) from 20 patients with PD was plated on blood agar and Pg colonies re-cultured to isolate individual strains of the bacterium. From each strain the PPAD gene was cloned, sequenced and analysed. The native PPAD gene in the reference Pg ATCC33277 strain was substituted with the polymorphic gene. The phenotype of clinical isolates harbouring polymorphism, the mutated and native ATCC33277 strains was examined. Further, periodontal clinical parameters were compared amongst patients infected by Pg expressing PPAD with and w/o polymorphism. Results A three amino acid polymorphism (G231N, E232T, N235D) was identified in the vicinity of the PPAD catalytic His residue in Pg isolates obtained from 30% of PD patients. The PPAD activity of clinical strains with polymorphism and the ATCC33277 mutant was 2-fold higher in comparison to the reference strain. Gingival fibroblasts infection with strains carrying polymorphic PPAD caused significantly higher upregulation of cyclooxygenase 2 (COX-2) than infection with native ATCC33277. Probing pocket depth (PPD) and clinical attachment level (CAL) assessment revealed that patients infected with Pg expressing polymorphic PPAD suffered from more severe disease than those carrying ‘classical’ Pg w/o polymorphism. Conclusions A three amino acid polymorphism of PPAD augments the virulence of Pg and severity of periodontitis apparently due to higher PPAD activity. The increased citrullination of bacterial and/or host proteins by Pg with the characterised PPAD genotype can trigger autoimmune response in RA. Acknowledgments National Science Centre, Poland (2012/07/B/NZ6/03524, K.G.), Foundation for Polish Science (TEAM, DPS/424-329/10, J.P.). References 1. Petersen PE, Bourgeois D, Ogawa H, et al.The global burden of oral diseases and risks to oral health. Bull World Health Organ 2003;83:661–69. 2. Eke PI, Dye BA, Wei L, et al.CDC Periodontal Disease Surveillance workgroup and representatives of the American Academy of Periodontology. Prevalence of periodontitis in adults in the United States: 2009 and 2010. J Dent Res 2012;91:914–20. 3. Socransky SS, Haffajee AD, Smith C, et al.Relation of counts of microbial species to clinical status at the sampled site. J Clin Periodontol 1991;18:766–75. 4. Holt SC, Ebersole J, Felton J, et al.Implantation of Bacteroides gingivalis in nonhuman primates initiates progression of periodontitis. Science 1998;239:55–57. 5. McGraw WT, Potempa J, Farley D, et al.Purification, characterisation, and sequence analysis of a potential virulence factor from Porphyromonas gingivalis, peptidylarginine deiminase. Infect Immun 1999;67:3248–56. 6. Rodriguez SB, Stitt BL, Ash DE.Expression of peptidylarginine deiminase from Porphyromonas gingivalis in Escherichia coli: enzyme purification and characterisation. Arch Biochem Biophys 2009;488:14–22. 7. Wegner N, Wait R, Sroka A, et al.Peptidylarginine deiminase from Porphyromonas gingivalis citrullinates human fibrinogen and α-enolase: implications for autoimmunity in rheumatoid arthritis. Arthritis Rheum 2010;62:2662–72. 8. Lundberg K, Kinloch A, Fisher BA, et al.Antibodies to citrullinated alpha-enolase peptide 1 are specific for rheumatoid arthritis and cross-react with bacterial enolase. Arthritis Rheum 2008;58:3009–19. 9. Quirke AM, Lugli EB, Wegner N, et al.Heightened immune response to autocitrullinated Porphyromonas gingivalis peptidylarginine deiminase: a potential mechanism for breaching immunologic tolerance in rheumatoid arthritis. Ann Rheum Dis 2014;73:263–69.

A3.03 Formation of novel citrullinated peptides by porphyromonasgingivalis pad enzyme: Implications for autoimmunity in rheumatoid arthritis

Background and objectives Peptidylarginine deiminase (PAD) enzymes are thought to be involved in rheumatoid arthritis (RA) due to presence of anti-citrullinated protein antibodies (ACPA). Periodontitis is a risk factor for RA, and periodontal pathogen Porphyromonas gingivalishas been proposed as the link due to expression of the unique prokaryotic PAD enzyme PPAD. PPAD has specificity for C-terminal arginine substrates, resulting in formation of citrullinated epitopes distinct from those formed by mammalian PADs, which could induce a citrulline specific response. By solving the 3D crystal structures of wild-type (wtPPAD) and inactive mutant PPAD (mutPPAD) we aimed to determine mechanisms for the unique activity of PPAD, and compare function to human PAD2 and PAD4 in vitro. Materials and methods Recombinant PAD2, PAD4 and wt-/mutPPAD were expressed and purified from E.coli. wt-/mutPPAD was crystallised with bound arginine ligands and the 3D structure solved using molecular replacement. In vitro, PPAD/PAD activity was measured using a colourimetric assay for detection of citrulline from synthetic peptides. PPAD/PAD specificity was then quantified by mass spectrometry of citrullinated peptides formed from fibrinogen, following cleavage by proteinase K or P.gingivalis protease arginine gingipain (RgpB). Results Structural information from wtPPAD and mutPPAD identified the key residues (R152, R154) in determining the C-terminal specificity of PPAD. Mutation of these residues significantly reduced enzyme activity. In vitro, PPAD citrullinates C-terminal arginines only, measured by both colourimetriccitrulline detection and mass spectrometry of citrullinated fibrinogen peptides. PAD2 and PAD4 produced 3 and 2.5 times more citrulline respectively from internal compared to C-terminal arginine residues from synthetic peptides. Sequencing of citrullinated fibrinogen peptides formed by PAD2 and PAD4 also showed higher occurrence of internally citrullinated peptides (65% and 25%), than C-terminal (15% and 4%). Conclusions PPAD has unique substrate specificity for C-terminal arginine, determined by residues R152 and R154. We have shown the peptides formed by PPAD are not formed readily by human PAD2 or PAD4 and thus would be unlikely to occur in vivo. Following P.gingivalis infection, production of novel citrullinated epitopes could be involved in the breach of tolerance to citrullinated peptides observed in RA.

IMMUNISATION WITH RECOMBINANT AUTOCITRULLINATED PORPHYROMONAS GINGIVALIS PEPTIDYLARGININE DEIMINASE INDUCES AUTOIMMUNITY TO ENOLASE AND ARTHRITIS IN DBA/1 MICE

Background and Objectives Rheumatoid arthritis (RA) is characterised by the presence of anti-citrullinated peptide antibodies (ACPA) years before disease onset. Increasing molecular and epidemiological evidence has linked periodontitis (PD) to RA. Porphyromonas gingivalis is unique amongst periodontal pathogens in possessing a citrullinating enzyme, peptidylarginine deiminase (PPAD) with the potential to generate citrullinated antigens driving the autoimmune response in RA. We have examined the immune response to several peptides/proteins of significance to RA in DBA/1 mice immunised with recombinant PPAD. Materials and Methods Twelve week old DBA/1 mice were immunised with one of two emulsions: 1) recombinant PPAD in complete Freund’s adjuvant (CFA) or 2) an inactive PPAD mutant (C351A) in CFA. Clinical score and paw swelling of mice (indicative of arthritis) recorded for ten days post onset. Antibody responses to PPAD and C351A, and a number of immunodominant ACPA target peptides: anti-citrullinated a-enolase peptide-1 (CEP1), vimentin (cVim), fibrinogen (cFib) and their uncitrullinated forms (REP-1, vim and fib) were examined in mouse serum using Enzyme-linked Immunosorbant assays (ELISAs). The Mann-Whitney U test was used to calculate p-values for differences between the sera groups for each antigen. Results By day 30 post immunisation, 20% of mice immunised with PPAD had developed arthritis-like swelling in their paws. There was no significant difference between the antibody response to PPAD and the antibody response to C351A in any of the mice tested. There was a significantly raised antibody response (p < 0.05) to both CEP1 and REP1 (mean 0.263; OD450) in the mice immunised with PPAD compared to the mice immunised with C351A (CEP1, mean 0.074 (OD450) and REP1 mean 0.150 (OD450). Antibody responses to cFib and Fib were similar in all mice, as were antibody responses to cVim and Vim. Conclusions The paw swelling and raised immune response to the immunodominant enolase peptide, both citrullinated (CEP1) and uncitrullinated (REP1), in mice immunised with autocitrullinated PPAD shows that PPAD induces arthritis and autoimmunity to enolase. This demonstrates that an active citrullinating PPAD can break tolerance to a major RA autoantigen and provides further molecular evidence linking P. gingivalis infection to RA.

CITRULLINATION IN HEALTHY AND INFLAMED LUNG TISSUE AS A PRIMING SITE FOR AUTOIMMUNITY IN RA

Background Anti-citrullinated peptide/protein antibodies (ACPAs) are the key pathogenic autoantibodies in rheumatoid arthritis (RA). Because smoking is a risk factor for RA and ACPA often appear years before the onset of disease, it has been proposed that the lung may be a site for priming the ACPA response. Previous studies using immunohistochemistry suggested that smoking upregulates the expression of PAD2 and PAD4 with the resultant increased expression of citrullinated proteins. However these studies are limited by the availability of healthy lung tissue. In this study we used lung tissue taken at a distance from the primary tumour in lobectomy specimens and antibody reactivity to PAD2 and PAD4 and to two important precursor antigens in RA, alpha-enolase and fibrinogen, was defined by immunoblotting to ensure specificity. Methods Lobectomy specimens from 40 subjects undergoing surgery for tumours or bronchiectasis (10 never smokers, 10 smokers without airflow limitation, 10 COPD ex-smokers and 10 COPD current smokers) were carefully dissected to remove a sample of uninvolved lung. The tissue samples were examined by immunoblotting with an anti-modified citrulline (AMC) antibody and scored for the level of citrullination with a semi-quantitative score from 0–3 by two blinded observers. The presence of PAD2, PAD4, alpha-enolase and fibrinogen was also determined by immunoblotting and scored. Recombinant proteins were used as positive controls and blots with secondary antibodies only were carried out to exclude non-specific cross-reactivity. Results Citrullinated proteins were detected in 37 out of the 40 lung tissue samples, including 9 out of 10 never smokers. The number of bands and intensity was slightly increased in the COPD smokers (mean score 1.7), followed by COPD ex-smokers (1.4), smokers (1.3) and never smokers without airflow limitation (1.1). PAD2 was detected in all samples, with the band intensity scores correlating roughly with those seen for citrullinated proteins i.e. highest amongst the COPD smokers (mean score 1.9) and lowest amongst never smokers (mean score 1.1). PAD4 and the RA antigens alpha-enolase and fibrinogen were observed in all lung tissue in comparable amounts regardless of disease and smoking status. There was also evidence of citrullination of alpha-enolase provided by co-migration of a ~50KD band recognised by AMC and an anti-enolase antibody, and the demonstration that alpha-enolase in lung tissue ran at several isoelectric spots by 2D electrophoresis. Conclusions We have shown that there is widespread citrullination of proteins in lung tissue from never smokers, and that there is a modest increase with smoking and COPD. This pattern of expression corresponds to that of PAD2. There is also expression of at least two major RA autoantigens and of PAD4. This supports the hypothesis that the lung is a site for priming the ACPA response, which is enhanced by smoking and and COPD.