Koen M. J. Janssen

Rheumatoid arthritis–associated autoantibodies in non–rheumatoid arthritis patients with mucosal inflammation: a case–control study

IntroductionRheumatoid arthritis–associated autoantibodies (RA-AAB) can be present in serum years before clinical onset of rheumatoid arthritis (RA). It has been hypothesized that initiation of RA-AAB generation occurs at inflamed mucosal surfaces, such as in the oral cavity or lungs. The aim of this study was to assess systemic presence of RA-AAB in patients without RA who had oral or lung mucosal inflammation.MethodsThe presence of RA-AAB (immunoglobulin A [IgA] and IgG anti-cyclic citrullinated peptide 2 antibodies (anti-CCP2), IgM and IgA rheumatoid factor (RF), IgG anti-carbamylated protein antibodies and IgG and IgA anti-citrullinated peptide antibodies against fibrinogen, vimentin and enolase) were determined in sera of non-RA patients with periodontitis (PD, n = 114), bronchiectasis (BR, n = 80) or cystic fibrosis (CF, n = 41). Serum RA-AAB levels were compared with those of periodontally healthy controls (n = 36). Patients with established RA (n = 86) served as a reference group. Association of the diseases with RA-AAB seropositivity was assessed with a logistic regression model, adjusted for age, sex and smoking.ResultsLogistic regression analysis revealed that IgG anti-CCP seropositivity was associated with BR and RA, whereas the association with PD was borderline significant. IgA anti-CCP seropositivity was associated with CF and RA. IgM RF seropositivity was associated with RA, whereas the association with BR was borderline significant. IgA RF seropositivity was associated with CF and RA. Apart from an influence of smoking on IgA RF in patients with RA, there was no influence of age, sex or smoking on the association of RA-AAB seropositivity with the diseases. Anti-CarP levels were increased only in patients with RA. The same held for IgG reactivity against all investigated citrullinated peptides.ConclusionAlthough overall levels were low, RA-AAB seropositivity was associated with lung mucosal inflammation (BR and CF) and may be associated with oral mucosal inflammation (PD). To further determine whether mucosal inflammation functions as a site for induction of RA-AAB and precedes RA, longitudinal studies are necessary in which RA-AAB of specifically the IgA isotype should be assessed in inflamed mucosal tissues and/or in their inflammatory exudates.

Antibodies against Porphyromonas gingivalis in seropositive arthralgia patients do not predict development of rheumatoid arthritis

Clinical studies point towards an association between periodontitis and rheumatoid arthritis (RA).1 ,2 A pathogenic role is suggested for Porphyromonas gingivalis .3 P gingivalis may contribute to the pathogenesis of RA by breaking immune tolerance through formation of (bacterial and human) citrullinated proteins, leading to anticitrullinated protein antibody production (ACPA).4 ,5 Since ACPA production precedes RA development6 and because P gingivalis IgG antibodies are long-term stable in untreated periodontitis patients,7 we investigated whether anti- P gingivalis antibody levels are prognostic for development of RA, by assessing these antibodies in a cohort of 289 adults at risk for RA. Patients with arthralgia and seropositivity for IgM-rheumatoid factor (IgM-RF) and/or ACPA were selected from a prospective follow-up study on arthritis development.8 They are further referred to as seropositive arthralgia patients (SAP); their median follow-up was 30 months (IQR 13–49). Baseline sera were used for measurement of ACPA, IgM-RF, C-reactive protein (CRP) and HLA-DRB1 SE carrier status.8 IgA, IgG and IgM antibody levels against P gingivalis were determined by in-house ELISA with a pooled lysate of clinical isolates of P gingivalis as …

Lessons to be learned from periodontitis

Purpose of reviewThis article reviews the link between periodontitis and rheumatoid arthritis (RA) with regard to similarities in genetic risk factors and immunopathogenesis. Emphasis is paid to the potential role of the periodontal pathogen Porphyromonas gingivalis in the etiopathogenesis of both periodontitis and RA, in particular by post-translational modification of arginine into citrulline. Recent findingsP. gingivalis, a major periodontal pathogen, is presently known as the only bacterium in the oral flora which contains a peptidyl arginine deiminase enzyme (PAD). This enzyme is necessary for citrullination. As a result, citrullinated proteins and P. gingivalis PAD, PAD2 and PAD4 (expressed by infiltrating neutrophils) are found in periodontal tissues. Autoantibodies directed to citrullinated proteins, so-called anticitrullinated protein antibodies (ACPAs), are found to be present in gingival crevicular fluid originating from inflamed gingival tissue. Furthermore, treatment studies have revealed that nonsurgical periodontal treatment, that is removal of sub-gingival calculus and biofilm deposits, is accompanied by a reduction in the severity of RA. SummaryIn this study the similarities in immune response and tissue degradation between RA and periodontitis are reviewed. It is shown that the two diseases share the same environmental and genetic risk factors, apart from the fact that there is a link between both diseases via citrullination of proteins by human PAD and P. gingivalis PAD.

Periodontitis and Rheumatoid Arthritis: What Do We Know?

BACKGROUND Currently, in the field of rheumatology, there is much attention given towards the possible causality between periodontitis and rheumatoid arthritis (RA), specifically regarding the role of Porphyromonas gingivalis (Pg). This bacterium is unique, having a citrullinating enzyme. Antibodies against citrullinated proteins are rather specific for RA. METHODS Because causality is ultimately tested in longitudinal cohort studies which currently do not exist for periodontitis and RA, this commentary applied Bradford Hill criteria on the existing literature to assess causality as the most likely interpretation of this association. CONCLUSIONS From an epidemiologic point of view, patients with RA have a higher incidence of periodontal disease than individuals without RA. In addition, there is a dose-response pattern in the association between the severity of periodontitis and RA disease activity. There are indications that periodontitis precedes RA, but there is no evidence yet available to show that Pg plays a direct role in this temporal relationship. The role of the unique characteristic of citrullination by Pg remains unexplained. However, in animal models, citrullination by Pg plays a distinct role in the development and aggravation of experimental arthritis. Although the role of Pg in RA remains speculative, a causative role for periodontitis as a chronic inflammatory disease caused by infectious agents in RA seems biologically plausible.

Commentary: Periodontitis and Rheumatoid Arthritis: What Do We Know?

BACKGROUND Currently, in the field of rheumatology, there is much attention given towards the possible causality between periodontitis and rheumatoid arthritis (RA), specifically regarding the role of Porphyromonas gingivalis (Pg). This bacterium is unique, having a citrullinating enzyme. Antibodies against citrullinated proteins are rather specific for RA. METHODS Because causality is ultimately tested in longitudinal cohort studies which currently do not exist for periodontitis and RA, this commentary applied Bradford Hill criteria on the existing literature to assess causality as the most likely interpretation of this association. CONCLUSIONS From an epidemiologic point of view, patients with RA have a higher incidence of periodontal disease than individuals without RA. In addition, there is a dose-response pattern in the association between the severity of periodontitis and RA disease activity. There are indications that periodontitis precedes RA, but there is no evidence yet available to show that Pg plays a direct role in this temporal relationship. The role of the unique characteristic of citrullination by Pg remains unexplained. However, in animal models, citrullination by Pg plays a distinct role in the development and aggravation of experimental arthritis. Although the role of Pg in RA remains speculative, a causative role for periodontitis as a chronic inflammatory disease caused by infectious agents in RA seems biologically plausible.

Regulatory CD4+T-Cell Subsets and Anti-Citrullinated Protein Antibody Repertoire: Potential Biomarkers for Arthritis Development in Seropositive Arthralgia Patients?

Objective Seropositive arthralgia patients (SAP) are at high risk of developing rheumatoid arthritis (RA). This prospective study aimed to determine whether altered peripheral regulatory T-cells (Tregs) and defined subsets, besides a broadened anti-citrullinated protein antibody (ACPA) response, may qualify as biomarkers for RA development in SAP. Methods Thirty-four consecutive SAP were prospectively assessed every 6 months for minimally 2 years. At inclusion, peripheral Treg (CD4+CD25+FoxP3+) numbers and subsets, defined as CD45RA+FoxP3low naive Tregs (Fr I), CD45RA-FoxP3high activated Tregs (Fr II) and CD45RA-FoxP3low non-Tregs (Fr III), were compared to age- and sex-matched healthy controls (HC, n = 16) and treatment-naive RA patients (n = 12). SAP that developed RA were compared to non-switchers and analyzed for Treg numbers and Treg subsets at inclusion. Also, Treg numbers and subsets were compared in switched SAP before and at diagnosis. To assess the ACPA repertoire, IgG and IgA reactivity was measured against citrullinated peptides from fibrinogen, α-enolase and vimentin. Results Treg numbers were similar between HC, SAP and RA patients. Although the bonafide Treg subsets Fr I and Fr II were comparable between groups, Fr III was increased in SAP compared to HC (p = 0.01). Fourteen (41%) SAP developed RA during follow-up. Their Treg numbers and subsets were comparable to non-switched SAP. At RA diagnosis, Treg numbers in switched SAP were similar to 6 months before. Switched SAP displayed a more diverse IgG ACPA repertoire compared to non-switched SAP (p = 0.046) and showed more IgA reactivity than non-switched SAP reaching significance for Fib1 only (p = 0.047). Conclusion Numbers of Total Treg and bonafide Treg subsets are not indicative for RA development in SAP, opposed to the ACPA repertoire.

PRESENCE OF SYSTEMIC ARTHRITIS AUTOANTIBODIES IN NON-RA PATIENTS WITH SEVERE PERIODONTITIS

Background Anti-citrullinated protein antibodies (ACPA), rheumatoid factor (RF) and anti-carbamylated proteins (anti-CarP) can be present before clinical manifestation of rheumatoid arthritis (RA). Periodontitis is regarded as a possible risk factor for RA, partly because of presence of citrullinated proteins in inflamed periodontal tissue. Porphyromonas gingivalis (PG), a periodontal pathogen that can citrullinate bacterial and human proteins is considered a possible contributor to disease onset of RA. Objectives To assess the systemic presence of ACPA, RF and anti-CarP in serum of severe periodontitis patients and periodontal healthy persons. Reactivity to citrullinated peptides was assessed in a subgroup. Methods In a group of 117 adult patients with severe periodontitis without other systemic disease (51±9.3 years, 58% female, 43% current smoker) and a group of 38 healthy controls with no signs of periodontitis (36±16 years, 53% female, 13% current smoker), the following serum autoantibodies were assessed by ELISA: IgG ACPA (anti-CCP2, Euro Diagnostica, cut off for positivity > 25U/ml), IgA, IgG and IgM RF (in-house, cut off for positivity > 25, >25 and >15 U/ml, respectively) and IgG anti-CarP (anti-carbamylated FCS and anti-carbamylated fibrinogen). IgA, IgG and IgM antibodies against periodontal pathogens PG and Prevotella intermedia (PI), which is closely related to PG, were also assessed. In a subpopulation of severe periodontitis patients (n=41) and periodontal healthy controls (n=13), IgG antibodies against citrullinated peptides were determined by ELISA. Extinctions were corrected for the native forms of the peptides and positive reactivity was defined as mean + 2 SD of the extinctions of periodontal healthy controls. Results Positivity for RF (IgM 6.0% (n=7), IgA 5.3% (n=6), IgG 1.7% (n=2)) and ACPA (1.7% (n=2)) was found in severe periodontitis patients, but in none of the periodontal healthy controls. Reactivity against citrullinated peptides was higher in severe periodontitis patients (positive for cit-fibrinogen-1 (n=2), for cit-fibrinogen-2 (n=7), for cit-fibrinogen-3 (n=5), for cit-enolase (n=7) and for cit-vimentin (n=6)) compared to periodontal healthy controls (positive only for cit-fibrinogen-2 (n=1)). Positivity for anti-CarP did not differ between periodontitis patients and HC. IgG anti-PG and anti-PI were significantly increased in periodontitis patients compared to periodontal healthy controls, but there was no correlation with RA auto-antibodies. Conclusions Positivity for RF, and ACPA, and reactivity against citrullinated peptides was found in patients with severe periodontitis but not in periodontal healthy persons. This observation suggests that to detect presence of ACPAs in an early stage, local ACPA production by B-cells in periodontium of periodontitis patients must be assessed. Disclosure of Interest None Declared

The combination of three autoantibodies, ACPA, RF and anti-CarP antibodies is highly specific for rheumatoid arthritis: implications for very early identification of individuals at risk to develop rheumatoid arthritis

In rheumatoid arthritis (RA), anti–citrullinated protein antibodies (ACPAs) and rheumatoid factor (RF) are commonly used to aid in the diagnosis. Although these autoantibodies are mainly found in RA, their specificity is not optimal. It is therefore difficult to identify RA patients, especially in very early disease, based on the presence of ACPAs and RF alone. In addition, anti–carbamylated protein (anti‐CarP) antibodies have diagnostic and prognostic value, since their presence is associated with joint damage in RA patients and also associated with the future development of RA in patients with arthralgia. Therefore, the aim of the present study was to investigate the value of combined antibody testing in relation to prediction and diagnosis of (early) RA.

AB0096 Presence of ACPA and RF Autoantibodies in Ulcerative Colitis and Crohn's Disease

Background Presence of arthritis autoantibodies is specific for rheumatoid arthritis (RA). Anti-citrullinated peptide antibodies (ACPA) and rheumatoid factor (RF) can be detected several years before the onset of arthritis and are thought to have a pathogenic role. However, where and when autoantibody production starts is unclear. A potential trigger could be chronic mucosal inflammation as in inflammatory bowel disease (IBD) since the gut microbiome is thought to be involved in induction of (auto) immunity. This assumption is supported by the demonstration of citrullinated proteins in intestinal tissue of IBD patients. Objectives To assess the prevalence of ACPA and RF in IBD patients as well as to assess the prevalence of joint complaints in autoantibody positive versus autoantibody negative IBD patients. Methods Serum was collected from 227 patients with ulcerative colitis (UC) and 164 with Crohns disease (CD) participating in the Dutch IBD pearl chain institute biobank initiative. Baseline disease characteristics including enthesitis, arthralgia, arthritis and comorbid conditions like spondylarthropathy were obtained from medical electronic records. IgG and IgA ACPA levels were measured by anti-CCP2 ELISA from EuroDiagnostica (ED) and IgM and IgA-RF levels were measured by in-house ELISA. The cut-off value for IgG ACPA positivity was 2 U/mL and for IgA ACPA 1.04 aU/ml based on the mean +2SD of a group of healthy reference samples. Seropositivity for IgM and IgA-RF was defined at ≥10 IU/ml and ≥25 IU/ml, respectively. Results Thirty-two (14.1%) UC and 33 (20.1%) CD patients had an IgG ACPA level >2 U/ml. However, all IgG ACPA levels were below the diagnostic cut off level for RA which is set at ≥25 U/ml. IgA ACPA levels were increased in 83 (36.5%) UC and 48 (29.3%) CD patients. IgM-RF seropositivity was observed in 11 (4.8%) UC and 6 (3.7%) CD patients. IgA-RF seropositivity was observed in 6 (2.6%) UC and 8 (4.9%) CD patients. A total of 101 (44.5%) UC and 76 (46.3%) CD were seropositive for at least 1 arthritis autoantibody. Arthralgias were reported in 19.1% of UC and 33.3% of CD, respectively. Among patients with arthralgias, the percentage of individuals being seropositive for 1 or more autoantibodies was comparable to patients without arthralgias. Conclusions The results of our study support the hypothesis that inflammation of intestinal mucosa induces low systemic levels of ACPA, which eventually may play a role in induction of RA. However, the development of arthralgias in IBD patients is independent on the presence of low systemic presence of arthritis autoantibodies. Disclosure of Interest None declared

SAT0035 Regulatory CD4+ T-Cell Levels and Anti Citrullinated Protein Antibody Repertoire as Biomarkers for Arthritis Development in Seropositive Arthralgia Patients

Background Early diagnosis and treatment of rheumatoid arthritis (RA) is of paramount importance for achieving a better disease outcome. Seropositive arthralgia patients (SAP), positive for anti citrullinated protein antibody (ACPA) and/or rheumatoid factor (RF) with (a history of) arthralgia, have a “high risk” of developing RA. Therefore it is necessary to understand which preclinical immunological factors play a role in the progression towards RA in SAP since this could help identify individuals earlier and result in early intervention. Objectives This prospective study aimed at identifying whether altered regulatory T-cells (Tregs) levels and subsets, besides a broadened ACPA response, are biomarkers for RA development in SAP. Methods Thirty-four consecutive SAP were prospectively assessed every 6 months for at least 2 years. Every visit, peripheral blood mononuclear cells (PBMCs) were isolated and stored. At inclusion, peripheral Treg (CD4+CD25+FoxP3+) levels and Treg subsets, defined as CD45RA+FoxP3low naive Treg cells (Fr I), CD45RA–FoxP3high activated Treg cells (Fr II) and cytokine producing CD45RA–FoxP3low non-Treg cells (Fr III), were compared to age and sex matched healthy controls (HC, n=16) and treatment naive RA patients (n=12). SAP that developed RA during follow-up were compared to non-switchers on Treg levels and functional Treg subsets. To assess broadening of the IgG and IgA ACPA repertoire in serum, reactivity was measured against citrullinated peptides from fibrinogen, α-enolase and vimentin. Results Total Treg levels were similar between HC, SAP and RA patients. Although functional Treg subsets Fr I and Fr II were comparable between groups, Fr III was increased in SAP compared to HC (p=0.01). Fourteen (41%) SAP developed RA during follow-up. Their Treg levels were comparable to those of non-switched SAP. At RA diagnosis, Treg levels in switched SAP were not changed compared to 6 months before. Switched SAP displayed an extended IgG ACPA repertoire compared to non-switched SAP (p=0.046). Conclusions Total and functional Treg levels do not indicate development of RA in SAP, in contrast to an extended IgG and IgA ACPA repertoire. Hence, there is presumably no contribution of altered Treg levels leading to the loss in suppression of autoimmunity in RA pathology. Disclosure of Interest None declared