Investigations of Porphyromonas gingivalis as a possible trigger of autoimmunity in the development of rheumatoid arthritis

Rheumatoid Arthritis (RA) is a severe autoimmune disease affecting 0.5-1% worldwide. Patients suffer from pain, disability, chronic joint inflammation, comorbidities and increased mortality. Although, the aetiology of RA is largely unknown, both genes and environment have been shown to contribute. The major risk factors of RA, smoking and HLA-DRB1 shared epitope (SE) alleles, associate primarily with the presence of autoantibodies to citrullinated proteins (ACPA). These antibodies are present in two thirds of patients, precede clinical symptoms, and predict a more destructive disease course. However, the exact trigger of ACPA production remains unclear. In addition to smoking, periodontitis (PD) has been epidemiologically linked to RA. Both smoking and PD cause local inflammation and increased protein citrullination. Moreover, Porphyromonas gingivalis (P.gin) - the keystone pathogen in chronic PD - is the only known prokaryote to express an enzyme that can citrullinate polypeptides (peptidyl arginine deiminase, denoted P.PAD). With this unique property, it was suggested that P.PAD could generate citrullinated epitopes in the inflamed periodontium, which would trigger a systemic ACPA response that eventually cause intraarticular inflammation. Based on this hypothesis, the overall aim of my PhD project was to investigate the role of Porphyromonas gingivalis in the aetiology of ACPA-positive RA, in terms of ACPA production, association with classical risk factors, and disease progression. Most studies in this thesis were performed in the population-based RA case-control cohort EIRA, where serum samples and information on genes (i.e. SE and PTPN22 polymorphism) and smoking history were available. In Study I, we showed that classical RA risk factors associated with specific ACPAs rather than the magnitude of the ACPA response, suggesting that the production of different ACPA fine-specificities is governed by partly different mechanisms. In Study II, we identified an association between anti-P.gin antibodies and RA (in particular ACPA-positive RA) that was even stronger than the association between smoking and RA. Moreover, we observed interactions between anti-P.gin antibodies and both SE and smoking in ACPA-positive RA. With Study III, we could demonstrate that anti-P.gin antibodies pre-dated clinical RA with up to 12 years. Study IV revealed a citrulline-specific antibody response to a P.PAD epitope in non-RA PD patients. Moreover, we identified a monoclonal antibody derived from RA blood, which exhibited cross-reactivity between citrullinated epitopes on bacterial (P.PAD) and human (vimentin) proteins. The data presented in this PhD thesis support a role for P.gin in the development of ACPApositive RA, and we propose that the pathway involves citrullination by P.PAD, followed by an antibody response, which cross-reacts with citrullinated human proteins, and that expansion of the autoimmune ACPA response in genetically susceptible individuals eventually triggers RA. It is my hope that the data presented herein can serve as a basis for disease-preventive strategies, as well as more detailed studies of disease mechanisms in RA aetiopathogenesis, ultimately aimed at the development of curative therapies

Genetic and environmental determinants for disease risk in subsets of rheumatoid arthritis defined by the anticitrullinated protein/peptide antibody fine specificity profile

OBJECTIVES: To increase understanding of the aetiology and pathogenesis of rheumatoid arthritis (RA), genetic and environmental risk factors for RA subsets, defined by the presence or absence of different anticitrullinated protein/peptide antibodies (ACPAs) targeting citrullinated peptides from α-enolase, vimentin, fibrinogen and collagen type II, were investigated. METHODS: 1985 patients with RA and 2252 matched controls from the EIRA case-control cohort were used in the study. Serum samples were assayed by ELISA for the presence of anticyclic citrullinated peptides (anti-CCP) antibodies and four different ACPA fine specificities. Cross-reactivity between ACPAs was examined by peptide absorption experiments. Genotyping was performed for HLA-DRB1 shared epitope (SE) alleles and the PTPN22 gene, while information regarding smoking was obtained by questionnaire. The association of genetic and environmental risk factors with different subsets of RA was calculated by logistic regression analysis. RESULTS: Limited cross-reactivity was observed between different ACPA fine specificities. In total, 17 RA subsets could be identified based on their different ACPA fine specificity profiles. Large differences in association with genetic and environmental determinants were observed between subsets. The strongest association of HLA-DRB1 SE, PTPN22 and smoking was identified for the RA subset which was defined by the presence of antibodies to citrullinated α-enolase and vimentin. CONCLUSION: This study provides the most comprehensive picture to date of how HLA-DRB1 SE, PTPN22 and smoking are associated with the presence of specific ACPA reactivities rather than anti-CCP levels. The new data will form a basis for molecular studies aimed at understanding disease development in serologically distinct subsets of RA.The Swedish Research CouncilVinnovaKing Gustaf V's 80-year foundationGums&Joints (FP7-Health-2010-261460)MasterSwitch (FP6-Health-2007-2.4.5-12)The Swedish Rheumatic FoundationThe Swedish Council for Working Life and Social ResearchThe IMI program BTCure (115142-2)Publishe

Drug Tolerant Anti-drug Antibody Assay for Infliximab Treatment in Clinical Practice Identifies Positive Cases Earlier

Publisher's version (útgefin grein)A subgroup of patients treated with infliximab lose response to the treatment and one reason for this is the development of anti-drug antibodies (ADA). If used optimally, measuring drug and ADA level could lead to a more personalized and efficient treatment regime, and enable identification of ADA-positive patients before the underlying disease flares or allergic reactions occur. With the use of a drug-tolerant ADA assay which can detect ADA irrespective of drug levels in the sample, we determined the impact of ADA on treatment failure to infliximab. The aims of this study were to estimate the real-life optimal serum infliximab (sIFX) level and set a clinical threshold value for a drug-tolerant ADA assay. Trough levels of sIFX were measured with ELISA. Free ADA was measured with two drug-sensitive methods (ELISA and a bioassay) and one drug-tolerant method (PandA). Two real-life cohorts treated with infliximab were included; a cross-sectional cohort including patients with inflammatory rheumatic diseases (n = 270) and a prospective cohort of rheumatoid arthritis (RA) patients (n = 73) followed for 1 year. Normal range of sIFX was estimated from the prospective cohort and an arbitrary optimal drug level was set to be between 1 and 6 μg/mL. Using this range, optimal sIFX was found in only 60% (163/270) of the patients in the cross-sectional cohort. These patients had significantly better treatment response than those with a drug level under 1 μg/mL, who had an ADA frequency of 34% (19/56) using the drug-tolerant method. In the prospective cohort, the drug-tolerant assay could identify 34% (53/155 samples) as ADA positive in samples with sIFX level >0.2 μg/mL. ADA were seldom detected in patients with >1 μg/mL sIFX, with three interesting exceptions. A clinically relevant ADA threshold was determined to be >3 RECL as measured with the drug-tolerant assay. In a real-life setting, there was a substantial number of patients with suboptimal drug levels and a proportion of these had ADA. Both too low and too high drug levels correlated with worse disease, but for different reasons. Adding a drug-tolerant assay enabled detection of ADA earlier and regardless of drug level at time of sampling.We would like to thank all patients for their participation in this study. We would also like to thank Consuelo Gomez, Pascual Gonzalez, Anna G. Mattsson, Arne St?hl, and Yousra Rehouma for excellent technical assistance. Funding. The research leading to these results has received support from Swelife, Stockholm County Council (ALF project) #20140333 and the Innovative Medicines Initiative Joint Undertaking under grant agreement no. 115303, resources of which are composed of financial contribution from the European Union's Seventh Framework Programme (FP7/2007-2013) and EFPIA companies' in kind contribution. This work was also supported by the grants from Aina (Ann) Wallstr?ms och Mary-Ann Sj?bloms Foundation for Medical Research, Professor Nanna Svartz Foundation, the Gothenburg Medical Society (GLS-889421 to RP), the Swedish Rheumatism Association (R-862061 and R-663511 to RP), Adlerbertska research Foundation and the Regional agreement on medical training and clinical research between the Western G?taland county council and the University of Gothenburg (ALFGBG-926621).Peer Reviewe

First exposure to rituximab is associated to high rate of anti-drug antibodies in systemic lupus erythematosus but not in ANCA-associated vasculitis

Background Anti-drug antibodies (ADAs) can impact on the efficacy and safety of biologicals, today used to treat several chronic inflammatory conditions. Specific patient groups may be more prone to develop ADAs. Rituximab is routinely used for ANCA-associated vasculitis (AAV) and as off-label therapy for systemic lupus erythematosus (SLE), but data on occurrence and predisposing factors to ADAs in these diseases is limited. Objectives To elucidate the rate of occurrence, and risk factors for ADAs against rituximab in SLE and AAV. Methods ADAs were detected using a bridging electrochemiluminescent (ECL) immunoassay in sera from rituximab-naive (AAV; n = 41 and SLE; n = 62) and rituximab-treated (AAV; n = 22 and SLE; n = 66) patients. Clinical data was retrieved from medical records. Disease activity was estimated by the SLE Disease Activity Index-2000 (SLEDAI-2 K) and the Birmingham Vasculitis Activity Score (BVAS). Results After first rituximab cycle, no AAV patients were ADA-positive compared to 37.8% of the SLE patients. Samples were obtained at a median (IQR) time of 5.5 (3.7-7.0) months (AAV), and 6.0 (5.0-7.0) months (SLE). ADA-positive SLE individuals were younger (34.0 (25.9-40.8) vs 44.3 (32.7-56.3) years, p = 0.002) and with more active disease (SLEDAI-2 K 14.0 (10.0-18.5) vs. 8.0 (6.0-14), p = 0.0017) and shorter disease duration (4.14 (1.18-10.08) vs 9.19 (5.71-16.93), p = 0.0097) compared to ADA-negative SLE. ADAs primarily occurred in nephritis patients, were associated with anti-dsDNA positivity but were not influenced by concomitant use of corticosteroids, cyclophosphamide or previous treatments. Despite overall reduction of SLEDAI-2 K (12.0 (7.0-16) to 4.0 (2.0-6.7), p &amp;lt; 0.0001), ADA-positive individuals still had higher SLEDAI-2 K (6.0 (4.0-9.0) vs 4.0 (2.0-6.0), p = 0.004) and their B cell count at 6 months follow-up was higher (CD19 + % 4.0 (0.5-10.0) vs 0.5 (0.4-1.0), p = 0.002). At retreatment, two ADA-positive SLE patients developed serum sickness (16.7%), and three had infusion reactions (25%) in contrast with one (5.2%) serum sickness in the ADA-negative group. Conclusions In contrast to AAV, ADAs were highly prevalent among rituximab-treated SLE patients already after the first course of treatment and were found to effect on both clinical and immunological responses. The high frequency in SLE may warrant implementations of ADA screening before retreatment and survey of immediate and late-onset infusion reactions.Funding Agencies|Region Stockholm by the ALF (Avtal for Lakarutbildning och Forskning); Swedish Rheumatism Association; Ingegerd Johanssons Fund; Karolinska Institutet FoundationsKarolinska Institutet; Swedish Kidney Foundation; Vinnova/Swelife [2017-04936]; Karolinska InstituteKarolinska Institutet; King Gustaf Vs 80-year Foundation</p

Rheumatoid Arthritis and Periodontal Disease: association between Salivary Citrulline, ACPA Levels and Clinical Presentation

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Concentration of antibodies against Porphyromonas gingivalis is increased before the onset of symptoms of rheumatoid arthritis

Background: The periodontal pathogen Porphyromonas gingivalis is hypothesized to be important in rheumatoid arthritis (RA) aetiology by inducing production of anti-citrullinated protein antibodies (ACPA). We have shown that ACPA precede RA onset by years, and that anti-P. gingivalis antibody levels are elevated in RA patients. The aim of this study was to investigate whether anti-P. gingivalis antibodies pre-date symptom onset and ACPA production. Methods: A case-control study (251 cases, 198 controls) was performed within the Biobank of Northern Sweden. Cases had donated blood samples (n = 422) before the onset of RA symptoms by 5.2 (6.2) years (median (interquartile range)). Blood was also collected from 192 RA patients following diagnosis. Antibodies against P. gingivalis virulence factor arginine gingipainB (RgpB), and a citrullinated peptide (CPP3) derived from the P. gingivalis peptidylarginine deiminase enzyme, were analysed by ELISA. Results: Anti-RgpB IgG levels were significantly increased in pre-symptomatic individuals (mean +/- SEM; 152.7 +/- 14.8 AU/ml) and in RA patients (114.4 +/- 16.9 AU/ml), compared with controls (p &lt; 0.001). Anti-CPP3 antibodies were detected in 5 % of pre-symptomatic individuals and in 8 % of RA patients, with elevated levels in both subsets (4.33 +/- 0.59 and 9.29 +/- 1.81 AU/ml, respectively) compared with controls (p &lt; 0.001). Anti-CPP3 antibodies followed the ACPA response, with increasing concentrations over time, whilst anti-RgpB antibodies were elevated and stable in the pre-symptomatic individuals with a trend towards lower levels after RA diagnosis. Conclusions: Anti-P. gingivalis antibody concentrations were significantly increased in RA patients compared with controls, and were detectable years before onset of symptoms of RA, supporting an aetiological role for P. gingivalis in the development of RA

Effects by periodontitis on pristane-induced arthritis in rats

Abstract Background An infection-immune association of periodontal disease with rheumatoid arthritis has been suggested. This study aimed to investigate the effect of pre-existing periodontitis on the development and the immune/inflammatory response of pristane-induced arthritis. Methods We investigated the effect of periodontitis induced by ligature placement and Porphyromonas gingivalis (P. gingivalis) infection, in combination with Fusobacterium nucleatum to promote its colonization, on the development of pristane-induced arthritis (PIA) in rats (Dark Agouti). Disease progression and severity of periodontitis and arthritis was monitored using clinical assessment, micro-computed tomography (micro-CT)/intraoral radiographs, antibody response, the inflammatory markers such as α-1-acid glycoprotein (α-1-AGP) and c-reactive protein (CRP) as well as cytokine multiplex profiling at different time intervals after induction. Results Experimentally induced periodontitis manifested clinically (P < 0.05) prior to pristane injection and progressed steadily until the end of experiments (15 weeks), as compared to the non-ligated arthritis group. Injection of pristane 8 weeks after periodontitis-induction led to severe arthritis in all rats demonstrating that the severity of arthritis was not affected by the pre-existence of periodontitis. Endpoint analysis showed that 89% of the periodontitis-affected animals were positive for antibodies against arginine gingipain B and furthermore, the plasma antibody levels to a citrullinated P. gingivalis peptidylarginine deiminase (PPAD) peptide (denoted CPP3) were significantly (P < 0.05) higher in periodontitis rats with PIA. Additionally, there was a trend towards increased pro-inflammatory and anti-inflammatory cytokine levels, and increased α-1-AGP levels in plasma from periodontitis-challenged PIA rats. Conclusions Pre-existence of periodontitis induced antibodies against citrullinated peptide derived from PPAD in rats with PIA. However, there were no differences in the development or severity of PIA between periodontitis challenged and periodontitis free rats