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.

BACKGROUND:Patients with atrial fibrillation (AF) often have sleep apnea (SA), but diagnosis of SA with polysomnography is costly. SA monitoring is a pacemaker feature that measures respiratory disturbance index, the sum of abnormal respiratory events divided by sleep duration. OBJECTIVE:The purpose of this study was to evaluate the incidence and severity of SA and its association with AF in an unselected population fitted with pacemakers. METHODS:RESPIRE (REgistry of Sleep APnea monItoring and Atrial Fibrillation in pacemakeR patients) was a multicenter, international, observational, open-label study following adult subjects for 18 months after implantation with an SA monitoring-enabled dual-chamber pacemaker. Severe SA was defined as average respiratory disturbance index ≥20 from implantation to follow-up visit. The first co-primary end point was the difference in significant AF (cumulative AF episodes lasting ≥24 hours over 2 consecutive days) between subjects with severe and those nonsevere SA at 12 months in the full analysis set (N = 553). The second co-primary end point was the rate of major serious adverse events at 18 months in the modified intention-to-treat set (N = 1024). RESULTS:Severe SA was detected in 31.1% (172 of 553). A higher incidence of significant AF was reported in patients with severe SA than in patients with nonsevere SA (25.0% vs 13.9%; difference 11.1%; 95% confidence interval 3.7%-18.4%; P = .002). Significant AF increased with time in both groups, but at a faster rate in the severe SA group. No intergroup difference in the overall rate of major serious adverse events was observed (P = .065). CONCLUSION:SA screening over 12 months identified severe SA in almost one-third of unselected patients fitted with pacemakers. Severe SA was associated with a higher incidence of significant AF.

Antibodies to citrullinated protein antigens (ACPAs) are part of the classification criteria for rheumatoid arthritis (RA) 1. ACPAs can be measured using generic tests, such as the cyclic citrullinated peptide (CCP) assays, that use artificially generated synthetic peptides with no homology to known proteins, which provide good diagnostic sensitivity and specificity. Peptides from real autoantigens (fine specificities) tend to be of lower diagnostic sensitivity and are used primarily for research into disease pathogenesis, rather than clinical management. In a recent study 2, we found 2 groups of ACPA fine specificities associated with different RA risk factors. One, dominated by antibodies to citrullinated α-enolase peptide 1 (CEP-1), was linked to smoking and shared epitope as previously described 3. The second group, linked to antibodies to a citrullinated peptide from tenascin-C (cTNC5), was associated with infection with the periodontal pathogen Prevotella intermedia. The relative distinctions between anti–CEP-1 and anti-cTNC5 prompted us to ask whether testing for these 2 fine specificities in combination with anti-CCP2 would be clinically useful in improving diagnosis or identifying clinical subsets among patients with RA. We reanalyzed previous data from 287 patients with RA and 330 osteoarthritis controls, and we retested all of the sera for anti–CEP-1 using a commercially available anti–CEP-1 enzyme-linked immunosorbent assay (Euroimmun), optimized for clinical use. The diagnostic sensitivity with anti–CEP-1 was increased from 39% 2 to 48%, with a specificity of 98%. We then examined whether the addition of anti–CEP-1 and anti-cTNC5 to CCP2 increased the overall diagnostic sensitivity. We identified 8 patients with RA who were negative for anti-CCP2 but positive for either or both of the fine specificities (Figure 1A), increasing the overall diagnostic sensitivity with ACPA from 84.5% obtained with anti-CCP2 alone to 87% if all 3 assays were combined. We predict that this modest increase in sensitivity could be an underestimate caused by ascertainment bias because anti-CCP2 positivity may have influenced the recruitment of patients to this cohort. In previous studies, using cohorts in which many of the patients were recruited before the CCP tests became available, anti–CEP-1 and/or anti-cTNC5 positivity was higher in the anti-CCP2–negative RA patients, increasing overall diagnostic sensitivity by ~5% 2-4. Therefore, the inclusion of these 2 additional fine specificities at the point of diagnosis could increase the sensitivity even further, though clearly this needs to be confirmed in prospective studies. We then investigated whether positivity for anti–CEP-1 and/or anti-cTNC5 identified distinct clinical subsets. For this analysis, we omitted the anti-CCP2–negative patients, as numbers were too small for statistical evaluation. We identified no difference in joint disease between patients who were anti-cTNC5 positive versus those who were anti–CEP-1 positive. However, when both fine specificities were combined with anti-CCP2, the 28-joint Disease Activity Score (DAS28) 5 in patients positive for all 3 markers was significantly increased (P = 0.038) compared to that in patients positive for only CCP2, with the difference in the Health Assessment Questionnaire score 6 approaching significance (P = 0.073). Triple-positive patients also had significantly higher levels of anti-CCP2 (P < 0.001) and rheumatoid factor (P < 0.001) (Figure 1B). This increase in disease severity with multiple antibody specificities is perhaps not surprising, as a similar phenomenon has long been known to occur in systemic lupus erythematosus (SLE) 7. In SLE, at least 6 different specificities would be routinely examined in every patient at presentation, with diagnosis established in the small proportion of antinuclear antibody–negative patients by the presence of anti-Ro antibodies. The same antibody also identifies rare but important clinical subsets: for example, an association with congenital heart block in babies of anti-Ro–positive mothers 7. In our patients with RA, no distinct clinical subsets were found, but we only examined joint-specific variables. It is possible that associations with extraarticular features may be recognized in future studies. Indeed, while anti–citrullinated vimentin antibodies are linked with bone loss 8, anti–citrullinated fibrinogen antibodies have been associated with atherosclerosis 9. For anti–CEP-1, a link with rheumatoid lung disease may be predicted by the association with smoking 3 and a high rate of positivity in patients with RA and bronchiectasis 10. Whether different ACPA fine specificities actively drive distinct aspects of joint disease and/or comorbidities also remains to be fully understood. We suggest that the only way to establish whether fine specificities are useful in improving diagnosis or identifying clinical subsets is to incorporate at least these 2 fine specificities into prospective studies so that in RA, multiple antibody assays become routine parts of clinical diagnosis and management. Supported by the European Community IMI (Innovative Medicines Initiative) project BTCure (grant 115142-2), the Kennedy Trust for Rheumatology Research (grant AZRYXS00), and a Veterans Affairs Clinical Science Research and Development Merit Award (CX000896). Dr. Mikuls’ work was supported by a grant from the NIH/National Institute of General Medical Sciences (U54-GM-115458). Dr. Midwood's work was supported by an Arthritis Research UK Senior Fellowship (grant 20003). Drs. Schwenzer, Venables, and Midwood have filed patents related to the diagnostic utility of cTNC5. Dr. Midwood is founder of, and consultant to, Nascient Ltd. Drs. Schwenzer and Quirke contributed equally to this work. All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Midwood had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Schwenzer, Quirke, Venables, Schlumberger, Payne, Mikuls, Midwood. Schwenzer, Quirke, Montgomery, Venables, Payne, Mikuls, Midwood. Schwenzer, Quirke, Venables, Sayles, Payne, Mikuls, Midwood.

Background. Lower socio-economic status (SES) has been associated with worse clinical outcomes, reduced functional ability and lower quality of life; however, little is known about the association between SES and the development of RA. A few studies have observed an inverse association between education level, a surrogate marker of SES, and risk of developing RA in Northern European populations. The objective was to investigate the association between SES on an individual level and risk of developing RA in a Southern European Mediterranean population.Methods. EPIC is a multicentre, pan-European prospective cohort study of apparently healthy populations. We undertook a nested case-control study to investigate risk factors for RA, by identifying incident RA cases (pre-RA) and matched controls amongst subjects enrolled in four EPIC cohorts in Italy and Spain. The lifestyle, environmental exposure, anthropometric information and blood samples were collected at baseline. Confirmed pre-RA cases were matched with controls by age, sex, centre, and date, time and fasting status at blood collection. The exposure was SES as measured by level of educational attainment categorised as university (referent), secondary school/technical/professional school, primary school completed, and none. The primary outcome was incident RA. Conditional logistic regression (CLR) analysis was adjusted for ACPA seropositivity, smoking status, and presence of shared epitope (SE). A further model also adjusted for other potential confounders, including body mass index (BMI), waist circumference, physical activity, and alcohol intake.Results. The …

Citrullination of proteins, a post-translational conversion of arginine residues to citrulline, is recognized in rheumatoid arthritis, but largely undocumented in cancer. Here we show that citrullination of the extracellular matrix by cancer cell derived peptidylarginine deiminase 4 (PAD4) is essential for the growth of liver metastases from colorectal cancer (CRC). Using proteomics, we demonstrate that liver metastases exhibit higher levels of citrullination and PAD4 than unaffected liver, primary CRC or adjacent colonic mucosa. Functional significance for citrullination in metastatic growth is evident in murine models where inhibition of citrullination substantially reduces liver metastatic burden. Additionally, citrullination of a key matrix component collagen type I promotes greater adhesion and decreased migration of CRC cells along with increased expression of characteristic epithelial markers, suggesting a role for citrullination in promoting mesenchymal-to-epithelial transition and liver metastasis. Overall, our study reveals the potential for PAD4-dependant citrullination to drive the progression of CRC liver metastasis.

Objectives To investigate the association between socioeconomic status (SES) on an individual level and incident RA Methods EPIC is a multicentre, pan-European prospective cohort study of apparently healthy populations. We undertook a nested case-control study to investigate risk factors for RA, by identifying incident RA cases (pre-RA) and matched controls amongst subjects enrolled in four EPIC cohorts in Italy and Spain. The lifestyle, environmental exposure, anthropometric information and blood samples were collected at baseline. Confirmed pre-RA cases were matched with controls by age, sex, centre, and date, time and fasting status at blood collection. The exposure was SES as measured by level of educational attainment categorised as university (referent), secondary school/technical/professional school, primary school completed, and none. The primary outcome was incident RA. Conditional logistic regression (CLR) analysis was adjusted for ACPA seropositivity, smoking status, and presence of shared epitope (SE). A further model also adjusted for other potential confounders, including body mass index (BMI), waist circumference, physical activity, and alcohol intake. Results The study sample included 398 individuals of which 99 individuals went on to subsequently develop RA. In this analysis, time to diagnosis (defined as time between date of blood sample and date of diagnosis), was 6.71 years (SD 3.43). A significant positive association was observed with level of educational attainment and RA incidence (secondary/technical vs university: OR 5.60, 95% CI 1.59–19.7, primary school vs university: OR 5.06, 95% CI 1.45–17.6, no education vs university: 7.11, 95% CI 1.37–36.8; p for trend 0.02) independent of ACPA seropositivity, SE and smoking). A significant positive association between level of educational attainment and RA incidence was confirmed in the fully adjusted model (secondary/technical vs university: OR 5.52, 95% CI 1.53–19.9, primary school vs university: OR 4.87, 95% CI 1.38–17.1, no education vs university: OR 6.48, 95% CI 1.21–34.6; p for trend 0.02). Conclusions Lower educational levels were independently associated with higher risk of incident RA in this European Mediterranean population. Disclosure of Interest None declared

Introduction Periodontitis (PD) is an environmental risk factor for rheumatoid arthritis (RA), and there is increasing evidence that this is due to infection with Porphyromonas gingivalis. P. gingivalis expresses 3 candidate enzymes potentially capable of breaking tolerance to citrullinated proteins. These include a unique prokaryotic peptidylarginine deiminase (PPAD), which in concert with arginine gingipain (Rgp) citrullinates C-terminal peptides from known RA autoantigens. The third enzyme, P.gingivalis enolase (PgEnolase), induces autoimmunity in mice potentially via molecular mimicry with conserved regions on mammalian enolase. The aim of this study was to determine the contribution of PPAD, PgEnolase and Rgp to the induction of autoimmunity to citrullinated proteins. Materials and methods Cultured P. gingivalis (W83) was separated into cellular subfractions and probed with anti-PPAD, anti-Rgp and anti-PgEnolase antibodies by immunoblotting, and using a citrulline specific probe. C57BL/6 mice were immunised without adjuvant with purified recombinant PPAD, Rgp and PgEnolase, alone or in combinations. Serum taken at 10 and 28 days was used to measure antibodies to PPAD, PgEnolase, and their immunodominant citrullinated peptides CPP3 and CPgE2. ACPA were measured by anti-CCP2, and anti-CEP-1 ELISA. Results PgEnolase, PPAD, and Rgp were detected in the outer membrane of the bacterium together with the highest concentration of citrullinated proteins. In mice, PPAD elicited a robust antibody response indicating it was highly immunogenic without adjuvant. Immunisation with all 3 enzymes (PPAD, Rgp and PgEnolase) induced a significantly increased antibody response to PgEnolase and citrullinated peptides CPP3 and CPgE2 compared to any other group (p<0.05). PgEnolase induced an ACPA response measured by anti-CCP2 and anti-CEP-1, which was increased with when co-immunised with PPAD and Rgp. Antibody responses were not citrulline specific indicated by the comparable level of antibodies reactive with arginine control peptides. None of the mice developed arthritis. Conclusion PPAD, Rgp and PgEnolase are localised in the P. gingivalis outer membrane and are available for immune surveillance. In combination in vivo, they induce anti-P. gingivalis antibodies seen in RA and ACPA. The lack of citrulline specificity of antibody responses is comparable to serology of PD and early pre-symptomatic RA. Thus our model could be used to study tolerance breakdown in PD and pre-RA.

ObjectiveIn 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.MethodsThe 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 β (cFIBβ) 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.ResultsA novel citrullinated peptide cCK13‐1 (444TSNASGR‐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, cFIBβ, and cVIM correlated with each other, and with smoking and shared epitope risk factors in RA.ConclusionThis 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.

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.

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.

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.

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.

Citrullination is catalysed by peptidylarginine deiminases (PADs) and may play a role in joint inflammation by generating proteins recognised by autoantibodies and/or by targeting histones and thereby modulating gene transcription. The PAD inhibitor Cl-amidine was shown to have a modest anti-inflammatory effect in collagen-induced arthritis (CIA), when given prophylactically at high doses. While much of this benefit was thought to be due to PAD4 inhibition, recent studies have highlighted the pro-inflammatory effects of PAD2. The objective of this study was to assess the therapeutic potential BB-Cl-amidine, which is 10 fold more active than Cl-amidine against PAD2. We chose CIA as a reproducible model of immune-mediated arthritis and used a clinically-relevant therapeutic protocol, in which mice were treated after disease onset. CIA was induced in DBA/1 mice by immunisation with bovine type II collagen. After disease onset, mice were treated daily with vehicle or BB-Cl-amidine (10 mg/kg, n = 12). On day 10, mice were culled, and paws, blood and lymph nodes collected for further analysis. In serum, cytokines were measured using a multiplex platform and anti-citrullinated peptide antibodies (ACPA) by ELISA. The phenotypes of T cells were determined by FACS. Citrullinated proteins were identified by mass spectrometry. Treatment of arthritic mice with BB-Cl-amidine, resulted in a significant reduction in clinical scores and paw swelling. Histological changes in joints were almost completely normalised by BB-Cl-amidine. Unexpectedly, while pro-inflammatory cytokine (TNF-α, IL-1β, IL-6) levels in serum remained unaffected by BB-Cl-amidine, IL-4, IL-5 and IL-10 levels were significantly elevated. In line with this, IL-4-expressing Th2 cells were increased in the lymph nodes of BB-Cl-amidine treated mice. Further analysis revealed a decrease in Th1 and Th17 numbers with BB-Cl-amidine. In addition BB-Cl-amidine reduced histone H3.2 citrullination, with little affect on the ACPA response which suggests that BB-CL-amidine may inhibit gene expression in disease via an epigenetic mechanism. In conclusion, BB-Cl-amidine is therapeutic in CIA due to immunomodulation rather than immunosuppression and supports anti-inflammatory Th2-type, while inhibiting pro-inflammatory Th1/Th17-type responses. We propose that these effects are mediated by transcriptional regulation and that targeting PADs is a realistic strategy for the treatment of chronic inflammatory diseases.

Early treatment of rheumatoid arthritis (RA) results in more effective disease suppression and can be key to a successful patient response. However, not all people who exhibit early synovitis develop RA; for example, in some, synovial inflammation resolves spontaneously.1 The factors that drive RA development remain unclear and clinical tools to predict RA development are imperfect. Tenascin-C is a proinflammatory matrix molecule that is absent from healthy joints but highly expressed in the joints of patients with RA.2 ,3 We identified an immunodominant peptide in citrullinated tenascin-C, cTNC5, antibodies against which are detected in around half of the patients with RA, and can be found years before disease onset in some individuals.4 Here, we sought to determine if anti-cTNC5 antibodies can discriminate among people with early synovial inflammation those who develop RA and those with other outcomes. Sera from 263 patients in the Birmingham early arthritis cohort were analysed. Patients were disease-modifying antirheumatic drug (DMARD) naive with clinically apparent synovitis of ≥1 joint and with inflammatory joint symptoms of ≤3 months’ duration. Patients were followed for 18 months to ensure development of full disease phenotype and to allow any resolving arthritis time to resolve. At 18 months, patients were assigned to the following outcome categories: persistent RA according to the American College of Rheumatology (ACR) 2010 criteria5 (RA, n=101), persistent non-RA arthritis (PNRA, n=66) and resolving arthritis (no clinically apparent joint swelling, no DMARD/steroid use in the previous 3 months, n=96). …

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.