Anne-Marie Quirke

Porphyromonas gingivalis Facilitates the Development and Progression of Destructive Arthritis through Its Unique Bacterial Peptidylarginine Deiminase (PAD)

Rheumatoid arthritis and periodontitis are two prevalent chronic inflammatory diseases in humans and are associated with each other both clinically and epidemiologically. Recent findings suggest a causative link between periodontal infection and rheumatoid arthritis via bacteria-dependent induction of a pathogenic autoimmune response to citrullinated epitopes. Here we showed that infection with viable periodontal pathogen Porphyromonas gingivalis strain W83 exacerbated collagen-induced arthritis (CIA) in a mouse model, as manifested by earlier onset, accelerated progression and enhanced severity of the disease, including significantly increased bone and cartilage destruction. The ability of P. gingivalis to augment CIA was dependent on the expression of a unique P. gingivalis peptidylarginine deiminase (PPAD), which converts arginine residues in proteins to citrulline. Infection with wild type P. gingivalis was responsible for significantly increased levels of autoantibodies to collagen type II and citrullinated epitopes as a PPAD-null mutant did not elicit similar host response. High level of citrullinated proteins was also detected at the site of infection with wild-type P. gingivalis. Together, these results suggest bacterial PAD as the mechanistic link between P. gingivalis periodontal infection and rheumatoid arthritis.

Influence of periodontal disease, Porphyromonas gingivalis and cigarette smoking on systemic anti-citrullinated peptide antibody titres.

BACKGROUND Anti-citrullinated protein antibody (ACPA) responses may precede clinical onset of rheumatoid arthritis. Porphyromonas gingivalis peptidylarginine deiminase can citrullinate proteins possibly inducing autoimmunity in susceptible individuals. AIM To determine whether periodontitis, carriage of P. gingivalis, smoking and periodontal therapy influence ACPA titres. METHODS Serum and plaque samples were collected from 39 periodontitis patients before and after non-surgical periodontal treatment, and from 36 healthy subjects. Carriage of P. gingivalis was determined by PCR of plaque DNA. ACPA was determined by anti-cyclic citrullinated peptide (CCP) enzyme-linked immunosorbent assay (ELISA). Anti-P. gingivalis titres were determined by ELISA. RESULTS Untreated periodontitis patients had higher anti-CCP antibody titres than healthy controls [three patients (8%) greater than manufacturer suggested assay diagnostic threshold (5 Assay Units/AU) versus none (0%); mean ± SEM: 1.37 ± 0.23 versus 0.40 ± 0.10 AU, p < 0.0001]. Periodontitis patients who smoked demonstrated lower anti-P. gingivalis (15956 ± 4385 versus 2512 ± 1290 Units/ml, p < 0.05), but similar anti-CCP than non-smoking periodontitis patients (smokers: 1.31 ± 0.35; non-smokers: 1.41 ± 0.32 AU). Healthy smokers demonstrated elevated anti-CCP titres (0.75 ± 0.19 AU), at levels between healthy non-smokers (0.15 ± 0.05 AU) and non-smoker periodontitis patients. Six months after periodontal treatment, there were significant reductions in anti-CCP (non-smokers p < 0.05) and anti-P. gingivalis (all participants p < 0.01). CONCLUSION In subjects with periodontitis, P. gingivalis infection may be responsible for inducing autoimmune responses that characterize rheumatoid arthritis.

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

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.

Citrullination of autoantigens: Upstream of TNFα in the pathogenesis of rheumatoid arthritis

The discovery of autoimmunity to citrullinated protein/peptide antigens (ACPA) has led the concept that ACPA may be the essential link between disease susceptibility factors and the production of TNFα, which ultimately accounts for the disease phenotype. In this review we will consider (1) the mechanisms of citrullination, both physiological and pathological, (2) how known genetic and environmental factors could drive this peculiar form of autoimmunity and (3) how the immune response could lead to excessive production of TNFα by the synovial cells and ultimately to the disease phenotype (Fig. 1).

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

PAD4-mediated citrullination of E2F-1 transcription factor and its interplay with acetylation affects inflammatory gene expression. 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.

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

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).

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.

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.

A1.5 Smoking is a risk factor for ACPA prior to onset of symptoms of rheumatoid arthritis in a cohort from southern europe.

Background and Objectives Rheumatoid arthritis (RA) is characterised by antibodies to citrullinated proteins (ACPA) which may be present several years before development of clinical symptoms. Smoking is a known risk factor for the development of RA, but data is scarce on the relationship of smoking with specific subsets of ACPA in the years before disease onset. In this study we examined the immune response to citrullinated antigens and a detailed smoking history, in a nested case-control study of subjects enrolled in the European Prospective Investigation into Cancer (EPIC) project who later developed RA. Materials and Methods The study sample included 412 participants from EPIC centres in Italy and Spain. Serum from 103 individuals who would eventually develop RA (pre-RA cases) were compared with three age-, sex- and centre-matched controls (n = 309). Antibodies to CCP, citrullinated-α-enolase peptide-1 (CEP-1; CKIHACitEIFDSCitGNPTVEC), citrullinated-fibrinogen (cFIB; CNEEGFFSACitGHRPLDKKC) and citrullinated-vimentin (cVIM; CVYATCitSSAVCitLCitSSVPC) were analysed by ELISA with positivity defined by 98th percentile of the controls. Results The median time to diagnosis in the pre-RA cases was 7 years (1.7–15.8 years). The frequency of positive antibodies (cases vs. controls) were: anti-CCP2 (20%; p < 0.001), anti-cFIB (18%; p < 0.001), anti-CEP-1 (15%; p < 0.001), and anti-cVIM (6%; p < 0.006). All of the antibodies increased in frequency closer to diagnosis and with no specificity being particularly prominent in very early presymptomatic autoimmunity. The frequency of ever smoking was higher amongst pre-RA cases compared with controls (59% vs. 47%, p = 0.02). Pre-RA cases who smoked had higher levels of ACPA by all measures, which reached statistical significance when comparing former smokers to other groups. Pre-RA former smokers were 2.5 times more likely to develop RA (OR 2.50, 95% CI: 1.28, 4.89; p = 0.007) and were four times more likely to have positive anti-CEP-1 (OR 4.06, 95% CI 1.02, 16.2) than pre-RA never smokers. Conclusions This is the first study of Pre-RA from a Southern European population in which we show that smoking is a risk factor not only for RA but for the development of presymptomatic autoimmunity, with citrullinated enolase being prominent. This strengthens the hypothesis that smoking is of aetiological importance in the very early stages of generating the autoantibodies that ultimately drive the disease.

A6.3 Patients with bronchiectasis, with or without rheumatoid arthritis, have an elevated anti-citrullinated peptide antibodies (ACPA) response.

Background and Objectives ACPA, particularly antibodies to citrullinated enolase and vimentin, are associated with smoking in patients with rheumatoid arthritis (RA). There is increasing evidence that these links are due to expression of citrullinated proteins in the lungs of smokers. Whether this is a direct effect of smoking or inflammation is presently poorly understood. Bronchiectasis, a strong risk factor for RA, tends to occur in non-smokers, and can therefore be used to examine the role of inflammation in generating an ACPA response independent of smoking. Materials and Methods This multi-centre study included 53 patients with HRCT proven bronchiectasis and RA, 50 patients with RA without lung disease, 124 bronchiectasis without RA and 87 asthma patients as controls. Serum samples were analysed for ACPAs using anti-CCP (2nd generation assay) and in-house ELISA’s were used to measure fine specificities to citrullinated α-enolase (CEP-1), citrullinated vimentin (cVim) and fibrinogen (cFib) together with their arginine-containing control peptides. Smoking history was detailed for all patients. Results Comparing the RA/bronchiectasis and the RA patients without lung disease, anti-CCP antibodies were more frequently positive (87% vs 48%) and titre significantly greater (p<0.0001) in the RA/bronchiectasis group, despite a lower frequency of smoking (42% vs 58%). The frequency and titre were also increased in the RA/bronchiectasis group for anti CEP-1 (60% vs 24%; p < 0.001), cVim (34% vs 11%; p < 0.001) and anti-cFib (38% vs 18%; p = 012). Comparing the bronchiectasis with asthma groups, none of who had RA, 5% vs 2% had anti-CCP, 9% vs 2% had anti CEP-1 and none were anti-cVim or anti-cFib positive. However, the increased ACPA response in bronchiectasis vs asthma was significant for anti-CEP-1 (p < 0.001). Testing with REP-1, the arginine-containing control peptide for enolase, showed that the response in the bronchiectasis patients was not citrulline-specific. Conclusions We have demonstrated that despite a low prevalence of smokers, RA/bronchiectasis patients have increased anti-CCP antibodies and an ACPA fine specificity profile similar to that previously shown in RA and smoking (anti-CEP-1 and anti-cVIM). Additionally we identified significantly elevated levels of anti-CEP-1 in the bronchiectatis patients, though unlike in the RA patients, the response was not citrulline-specific, similar to that previously reported for periodontitis. This indicates that the lung may be an initiating site for immunity to RA autoantigens due to inflammation independent of smoking.