cIAP1/2 inhibition synergizes with TNF inhibition in autoimmunity by down-regulating IL-17A and inducing Tregs.

IL-17 and TNF-α are major effector cytokines in chronic inflammation. TNF-α inhibitors have revolutionized the treatment of rheumatoid arthritis (RA), although not all patients respond, and most relapse after treatment withdrawal. This may be due to a paradoxical exacerbation of TH17 responses by TNF-α inhibition. We examined the therapeutic potential of targeting cellular inhibitors of apoptosis 1 and 2 (cIAP1/2) in inflammation by its influence on human TH subsets and mice with collagen-induced arthritis. Inhibition of cIAP1/2 abrogated CD4+ IL-17A differentiation and IL-17 production. This was a direct effect on T cells, mediated by reducing NFATc1 expression. In mice, cIAP1/2 inhibition, when combined with etanercept, abrogated disease activity, which was associated with an increase in Tregs and was sustained after therapy retraction. We reveal an unexpected role for cIAP1/2 in regulating the balance between TH17 and Tregs and suggest that combined therapeutic inhibition could induce long-term remission in inflammatory diseases.

High erythrocyte levels of the n-6 polyunsaturated fatty acid linoleic acid are associated with lower risk of subsequent rheumatoid arthritis in a southern European nested case-control study.

OBJECTIVES: Findings relating to dietary intake of n-3 polyunsaturated fatty acids (PUFA) and risk of rheumatoid arthritis (RA) are mixed. Erythrocyte membrane PUFA is an accurate objective biomarker of PUFA status; however, there are little data on erythrocyte membrane PUFA and risk of RA. The objective was therefore to compare erythrocyte membrane PUFA between pre-RA individuals and matched controls from a population-based sample, and specifically to test the hypothesis that higher levels of longer chain n-3 PUFA are associated with lower risk of RA. METHODS: The European Prospective Investigation into Cancer and Nutrition (EPIC) is a large European prospective cohort study of apparently healthy populations. We undertook a nested case-control study by identifying RA cases with onset after enrolment (pre-RA) in four EPIC cohorts in Italy and Spain. Confirmed pre-RA cases were matched with controls by age, sex, centre, and date, time and fasting status at blood collection. Conditional logistic regression analysis was used to estimate associations of PUFA with the development of RA, adjusting for potential confounders including body mass index, waist circumference, education level, physical activity, smoking status and alcohol intake. RESULTS: The study analysed samples from 96 pre-RA subjects and 258 matched controls. In this analysis, the median time to diagnosis (defined as time between date of blood sample and date of diagnosis) was 6.71 years (range 0.8-15). A significant inverse association was observed with n-6 PUFA linoleic acid (LA) levels and pre-RA in the fully adjusted model (highest tertile: OR 0.29; 95% CI 0.12 to 0.75; P for trend 0.01). No association was observed with any individual n-3 PUFA, total n-3 PUFA or total n-3:n-6 ratio. CONCLUSIONS: Erythrocyte levels of the n-6 PUFA LA were inversely associated with risk of RA, whereas no associations were observed for other n-6 or n-3 PUFA. Further work is warranted to replicate these findings and to investigate if lower LA levels are a bystander or contributor to the process of RA development.

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.

OBJECTIVE: 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. METHODS: The citrullinomes of GCF and periodontal tissue from patients with PD were mapped by mass spectrometry. ACPAs of CK13 (cCK13), tenascin-C (cTNC5), vimentin (cVIM), α-enolase (CEP-1), and fibrinogen ß (cFIBß) were examined by enzyme-linked immunosorbent assay in patients with RA (n = 287) and patients with osteoarthritis (n = 330), and cross-reactivity was assessed by inhibition assays. RESULTS: A novel citrullinated peptide cCK13-1 (444 TSNASGR-Cit-TSDV-Cit-RP458 ) identified in GCF exhibited elevated antibody responses in RA patients (24%). Anti-cCK13-1 antibody levels correlated with anti-cTNC5 antibody levels, 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. Levels of antibodies to CEP-1, cFIBß, and cVIM correlated with each other, and with smoking and shared epitope risk factors in RA. CONCLUSION: This study identifies 2 groups of ACPA fine specificities associated with different RA risk factors. One is predominantly linked to smoking and shared epitope, and the other links anti-cTNC5 and cCK13-1 to infection with the periodontal pathogen P intermedia.

Abrogation of collagen-induced arthritis by a peptidyl arginine deiminase inhibitor is associated with modulation of T cell-mediated immune responses.

Proteins containing citrulline, a post-translational modification of arginine, are generated by peptidyl arginine deiminases (PAD). Citrullinated proteins have pro-inflammatory effects in both innate and adaptive immune responses. Here, we examine the therapeutic effects in collagen-induced arthritis of the second generation PAD inhibitor, BB-Cl-amidine. Treatment after disease onset resulted in the reversal of clinical and histological changes of arthritis, associated with a marked reduction in citrullinated proteins in lymph nodes. There was little overall change in antibodies to collagen or antibodies to citrullinated peptides, but a shift from pro-inflammatory Th1 and Th17-type responses to pro-resolution Th2-type responses was demonstrated by serum cytokines and antibody subtypes. In lymph node cells from the arthritic mice treated with BB-Cl-amidine, there was a decrease in total cell numbers but an increase in the proportion of Th2 cells. BB-Cl-amidine had a pro-apoptotic effect on all Th subsets in vitro with Th17 cells appearing to be the most sensitive. We suggest that these immunoregulatory effects of PAD inhibition in CIA are complex, but primarily mediated by transcriptional regulation. We suggest that targeting PADs is a promising strategy for the treatment of chronic inflammatory disease.

Citrullination-acetylation interplay guides E2F-1 activity during the inflammatory response.

Peptidyl arginine deiminase 4 (PAD4) is a nuclear enzyme that converts arginine residues to citrulline. Although increasingly implicated in inflammatory disease and cancer, the mechanism of action of PAD4 and its functionally relevant pathways remains unclear. E2F transcription factors are a family of master regulators that coordinate gene expression during cellular proliferation and diverse cell fates. We show that E2F-1 is citrullinated by PAD4 in inflammatory cells. Citrullination of E2F-1 assists its chromatin association, specifically to cytokine genes in granulocyte cells. Mechanistically, citrullination augments binding of the BET (bromodomain and extra-terminal domain) family bromodomain reader BRD4 (bromodomain-containing protein 4) to an acetylated domain in E2F-1, and PAD4 and BRD4 coexist with E2F-1 on cytokine gene promoters. Accordingly, the combined inhibition of PAD4 and BRD4 disrupts the chromatin-bound complex and suppresses cytokine gene expression. In the murine collagen-induced arthritis model, chromatin-bound E2F-1 in inflammatory cells and consequent cytokine expression are diminished upon small-molecule inhibition of PAD4 and BRD4, and the combined treatment is clinically efficacious in preventing disease progression. Our results shed light on a new transcription-based mechanism that mediates the inflammatory effect of PAD4 and establish the interplay between citrullination and acetylation in the control of E2F-1 as a regulatory interface for driving inflammatory gene expression.

Antibodies to Porphyromonas gingivalis Indicate Interaction Between Oral Infection, Smoking, and Risk Genes in Rheumatoid Arthritis Etiology.

OBJECTIVE: To investigate the role of the periodontal pathogen Porphyromonas gingivalis in the etiology of rheumatoid arthritis (RA) by analyzing the antibody response to the P gingivalis virulence factor arginine gingipain type B (RgpB) in relation to anti-citrullinated protein antibodies (ACPAs), smoking, and HLA-DRB1 shared epitope (SE) alleles in patients with periodontitis, patients with RA, and controls. METHODS: Anti-RgpB IgG was measured by enzyme-linked immunosorbent assay in 65 periodontitis patients and 59 controls without periodontitis, and in 1,974 RA patients and 377 controls without RA from the Swedish population-based case-control Epidemiological Investigation of Rheumatoid Arthritis (EIRA) study. Autoantibody status, smoking habits, and genetic data were retrieved from the EIRA database. Differences in antibody levels were examined using the Mann-Whitney U test. Unconditional logistic regression was used to calculate odds ratios (ORs) with 95% confidence intervals (95% CIs) for the association of anti-RgpB IgG with different subsets of RA patients. RESULTS: Anti-RgpB antibody levels were significantly elevated in periodontitis patients compared to controls without periodontitis, in RA patients compared to controls without RA, and in ACPA-positive RA patients compared to ACPA-negative RA patients. There was a significant association between anti-RgpB IgG and RA (OR 2.96 [95% CI 2.00, 4.37]), which was even stronger than the association between smoking and RA (OR 1.37 [95% CI 1.07, 1.74]), and in ACPA-positive RA there were interactions between anti-RgpB antibodies and both smoking and the HLA-DRB1 SE. CONCLUSION: Our study suggests that the previously reported link between periodontitis and RA could be accounted for by P gingivalis infection, and we conclude that P gingivalis is a credible candidate for triggering and/or driving autoimmunity and autoimmune disease in a subset of RA patients.

Citrullination in the periodontium--a possible link between periodontitis and rheumatoid arthritis.

OBJECTIVES: The aim of the present study was to assess human and bacterial peptidylarginine deiminase (PAD) activity in the gingival crevicular fluid (GCF) in the context of serum levels of antibodies against citrullinated epitopes in rheumatoid arthritis and periodontitis. MATERIALS AND METHODS: Human PAD and Porphyromonas gingivalis-derived enzyme (PPAD) activities were measured in the GCF of 52 rheumatoid arthritis (RA) patients (48 with periodontitis and 4 without) and 44 non-RA controls (28 with periodontitis and 16 without). Serum antibodies against citrullinated epitopes were measured by ELISA. Bacteria being associated with periodontitis were determined by nucleic-acid-based methods. RESULTS: Citrullination was present in 26 (50%) RA patients and 23 (48%) controls. PAD and PPAD activities were detected in 36 (69%) and 30 (58%) RA patients, respectively, and in 30 (68%) and 21 (50%) controls, respectively. PPAD activity was higher in RA and non-RA patients with periodontitis than in those without (p = 0.038; p = 0.004), and was detected in 35 of 59 P. gingivalis-positive samples, and in 16 of 37 P. gingivalis-negative samples in association with high antibody levels against that species. CONCLUSIONS: PAD and PPAD activities within the periodontium are elevated in RA and non-RA patients with periodontitis. PPAD secreted by P. gingivalis residing in epithelial cells may exert its citrullinating activity in distant regions of the periodontium or even distant tissues. CLINICAL RELEVANCE: In periodontitis, the citrullination of proteins/peptides by human and bacterial peptidylarginine deiminases may generate antibodies after breaching immunotolerance in susceptible individuals.

Identification of an immunodominant peptide from citrullinated tenascin-C as a major target for autoantibodies in rheumatoid arthritis.

OBJECTIVES: We investigated whether citrullinated tenascin-C (cTNC), an extracellular matrix protein expressed at high levels in the joints of patients with rheumatoid arthritis (RA), is a target for the autoantibodies in RA. METHODS: Citrullinated sites were mapped by mass spectrometry in the fibrinogen-like globe (FBG) domain of tenascin-C treated with peptidylarginine deiminases (PAD) 2 and 4. Antibodies to cyclic peptides containing citrullinated sites were screened in sera from patients with RA by ELISA. Potential cross-reactivity with well-established anticitrullinated protein antibody (ACPA) epitopes was tested by inhibition assays. The autoantibody response to one immunodominant cTNC peptide was then analysed in 101 pre-RA sera (median 7 years before onset) and two large independent RA cohorts. RESULTS: Nine arginine residues within FBG were citrullinated by PAD2 and PAD4. Two immunodominant peptides cTNC1 (VFLRRKNG-cit-ENFYQNW) and cTNC5 (EHSIQFAEMKL-cit-PSNF-cit-NLEG-cit-cit-KR) were identified. Antibodies to both showed limited cross-reactivity with ACPA epitopes from α-enolase, vimentin and fibrinogen, and no reactivity with citrullinated fibrinogen peptides sharing sequence homology with FBG. cTNC5 antibodies were detected in 18% of pre-RA sera, and in 47% of 1985 Swedish patients with RA and 51% of 287 North American patients with RA. The specificity was 98% compared with 160 healthy controls and 330 patients with osteoarthritis. CONCLUSIONS: There are multiple citrullination sites in the FBG domain of tenascin-C. Among these, one epitope is recognised by autoantibodies that are detected years before disease onset, and which may serve as a useful biomarker to identify ACPA-positive patients with high sensitivity and specificity in established disease.

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 PPAD(C351A) 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.

Smoking, Porphyromonas gingivalis and the immune response to citrullinated autoantigens before the clinical onset of rheumatoid arthritis in a Southern European nested case-control study.

BACKGROUND: Antibodies to citrullinated proteins (ACPA) occur years before RA diagnosis. Porphyromonas gingivalis expresses its own peptidylarginine deiminase (PPAD), and is a proposed aetiological factor for the ACPA response. Smoking is a risk factor for both ACPA-positive RA and periodontitis. We aimed to study the relation of these factors to the risk of RA in a prospective cohort. METHODS: We performed a nested case-control study by identifying pre-RA cases in four populations from the European Prospective Investigation into Cancer and nutrition, matched with three controls. Data on smoking and other covariates were obtained from baseline questionnaires. Antibodies to CCP2 and citrullinated peptides from α-enolase, fibrinogen, vimentin and PPAD were measured. Antibodies to arginine gingipain (RgpB) were used as a marker for P.gingivalis infection and validated in a separate cohort of healthy controls and subjects with periodontitis. RESULTS: We studied 103 pre-RA cases. RA development was associated with several ACPA specificities, but not with antibodies to citrullinated PPAD peptides. Antibody levels to RgpB and PPAD peptides were higher in smokers but were not associated with risk of RA or with pre-RA autoimmunity. Former but not current smoking was associated with antibodies to α-enolase (OR 4.06; 95 % CI 1.02, 16.2 versus 0.54; 0.09-3.73) and fibrinogen peptides (OR 4.24; 95 % CI 1.2-14.96 versus 0.58; 0.13-2.70), and later development of RA (OR 2.48; 95 % CI 1.27-4.84 versus 1.57; 0.85-2.93), independent of smoking intensity. CONCLUSIONS: Smoking remains a risk factor for RA well before the clinical onset of disease. In this cohort, P.gingivalis is not associated with pre-RA autoimmunity or risk of RA in an early phase before disease-onset. Antibodies to PPAD peptides are not an early feature of ACPA ontogeny.

Bronchiectasis is a Model for Chronic Bacterial Infection Inducing Autoimmunity in Rheumatoid Arthritis.

OBJECTIVE: To examine the potential of chronic severe bacterial infection to generate rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPAs), by studying patients with bronchiectasis (BR) alone and BR patients with rheumatoid arthritis (BR/RA). METHODS: We studied 122 patients with BR alone, 50 patients with BR/RA, and 50 RA patients without lung disease, as well as 87 patients with asthma and 79 healthy subjects as controls. RF levels were measured using an automated analyzer, and cyclic citrullinated peptide 2 (CCP-2) was used to detect ACPAs. The fine specificities of citrullinated α-enolase peptide 1 (CEP-1), Cit-vimentin, and Cit-fibrinogen to their arginine-containing control peptides (arginine-containing α-enolase peptide 1 [REP-1], vimentin, and fibrinogen) were measured by enzyme-linked immunosorbent assay. RESULTS: Among the BR patients and control subjects, 39% and 42%, respectively, were ever-smokers. The frequency of RF positivity in serum was increased in BR patients compared with controls (25% versus 10%), as were the frequencies of antibodies to CCP-2 (5% versus 0%), CEP-1 (7% versus 4%), Cit-vimentin (7% versus 4%), and Cit-fibrinogen (12% versus 4%), although only the differences for RF and Cit-fibrinogen were significant (P < 0.05). We observed a corresponding increase in the frequency of antibodies to the arginine-containing control peptides in BR patients compared with controls (for REP-1, 19% versus 4% [P < 0.01]; for vimentin, 16% versus 4% [P < 0.05]), demonstrating that the ACPA response in patients with BR is not citrulline specific. The lack of citrulline specificity was further confirmed by absorption studies. In BR/RA patients, all ACPA responses were highly citrulline specific. CONCLUSION: Bronchiectasis is an unusual but potent model for the induction of autoimmunity in RA by bacterial infection in the lung. Our study suggests that the ACPA response is not citrulline specific during the early stages of tolerance breakdown but becomes more specific in patients with BR in whom BR/RA develops.

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

INTRODUCTION: Smoking 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). METHODS: Lung 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. RESULTS: Within 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. CONCLUSIONS: We 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.

The autoantibody repertoire in periodontitis: a role in the induction of autoimmunity to citrullinated proteins in rheumatoid arthritis?

BACKGROUND: Studies suggest that periodontitis may be a risk factor for rheumatoid arthritis (RA). The purpose of this study was to determine whether periodontitis is associated with autoantibodies characteristic of RA. METHODS: Serum samples were tested for anti-cyclic citrullinated peptide (CCP), anti-mutated citrullinated vimentin (MCV), anti-citrullinated α-enolase peptide-1 (CEP-1), anti-citrullinated vimentin (cit-vim), anti-citrullinated fibrinogen (cit-fib) and their uncitrullinated forms anti-CParg (negative control for anti-CCP), anti-arginine-containing α-enolase peptide-1 (REP-1), anti-vimentin and anti-fibrinogen antibodies in patients with and without periodontitis, none of whom had RA. RESULTS: Periodontitis, compared with non-periodontitis, was associated with a normal frequency of anti-CCP and anti-MCV (∼1%) but a higher frequency of positive anti-CEP-1 (12% vs 3%; p=0.02) and its uncitrullinated form anti-REP-1 (16% vs 2%; p<0.001). Positive antibodies against uncitrullinated fibrinogen and CParg were also more common among those with periodontitis compared to non-periodontitis patients (26% vs 3%; p<0.001, and 9% vs 3%; p=0.06). After adjusting for confounders, patients with periodontitis had 43% (p=0.03), 71% (p=0.002) and 114% (p<0.001) higher anti-CEP-1, anti-REP-1 and anti-fibrinogen titres, compared with non-periodontitis. Non-smokers with periodontitis, compared with non-periodontitis, had significantly higher titres of anti-CEP-1 (103%, p<0.001), anti-REP-1 (91%, p=0.001), anti-vimentin (87%, p=0.002), and anti-fibrinogen (124%, p<0.001), independent of confounders, confirming that the autoantibody response in periodontitis was not due to smoking. CONCLUSIONS: We have shown that the antibody response in periodontitis is predominantly directed to the uncitrullinated peptides of the RA autoantigens examined in this study. We propose that this loss of tolerance could then lead to epitope spreading to citrullinated epitopes as the autoimmune response in periodontitis evolves into that of presymptomatic RA.

Smoking, the HLA-DRB1 shared epitope and ACPA fine-specificity in Koreans with rheumatoid arthritis: evidence for more than one pathogenic pathway linking smoking to disease.

OBJECTIVES: Data from North European rheumatoid arthritis (RA) populations has suggested a particularly strong association of gene-environment interaction between smoking and HLA-DRB1 shared epitope (SE) with antibodies to citrullinated α-enolase (CEP-1) and vimentin (cVim) peptides. We investigated this further by examining anticitrullinated peptide/protein antibody (ACPA) fine specificity in a Korean cohort, where there are notable differences in the RA-associated HLA-DRB1 alleles. METHODS: Antibodies to fibrinogen (cFib), α-enolase (CEP-1) and vimentin (cVim) peptides and cyclic citrullinated peptide (CCP) were measured in 513 cases. The Mann-Whitney U test was used to compare antibody levels. Logistic regression generated ORs for RA in a case-control analysis with 1101 controls. Association of ACPA status and erosion in patients with RA was examined by logistic regression. RESULTS: Anti-CCP, CEP-1, cVim and fibrinogen peptides were found in 86.7%, 63.9%, 45.5% and 74.7%, respectively. The number of ACPA and their levels were associated with SE, with evidence of a gene-dosage effect. There was a particular association of smoking with levels of anti-CEP-1. However, a gene-environment interaction was associated with all the ACPA positive subgroups, albeit the highest OR was seen with the anti-CCP+/cVim+ subset. In the absence of SE, smoking only conferred risk for anti-CCP negative subsets. The presence of erosions was not associated with the number of positive ACPA or specificity. CONCLUSIONS: The SE governed the magnitude and diversity of the ACPA response, but its interaction with smoking did not exclusively segregate with any of the ACPA specificities studied here. Smoking was associated with RA by SE-dependent and independent effects.

Heightened immune response to autocitrullinated Porphyromonas gingivalis peptidylarginine deiminase: a potential mechanism for breaching immunologic tolerance in rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is characterised by autoimmunity to citrullinated proteins, and there is increasing epidemiologic evidence linking Porphyromonas gingivalis to RA. P gingivalis is apparently unique among periodontal pathogens in possessing a citrullinating enzyme, peptidylarginine deiminase (PPAD) with the potential to generate antigens driving the autoimmune response. OBJECTIVES: To examine the immune response to PPAD in patients with RA, individuals with periodontitis (PD) and controls (without arthritis), confirm PPAD autocitrullination and identify the modified arginine residues. METHODS: PPAD and an inactivated mutant (C351A) were cloned and expressed and autocitrullination of both examined by immunoblotting and mass spectrometry. ELISAs using PPAD, C351A and another P gingivalis protein arginine gingipain (RgpB) were developed and antibody reactivities examined in patients with RA (n=80), individuals with PD (n=44) and controls (n=82). RESULTS: Recombinant PPAD was a potent citrullinating enzyme. Antibodies to PPAD, but not to Rgp, were elevated in the RA sera (median 122 U/ml) compared with controls (median 70 U/ml; p<0.05) and PD (median 60 U/ml; p<0.01). Specificity of the anti-peptidyl citrullinated PPAD response was confirmed by the reaction of RA sera with multiple epitopes tested with synthetic citrullinated peptides spanning the PPAD molecule. The elevated antibody response to PPAD was abolished in RA sera if the C351A mutant was used on ELISA. CONCLUSIONS: The peptidyl citrulline-specific immune response to PPAD supports the hypothesis that, as a bacterial protein, it might break tolerance in RA, and could be a target for therapy.