ObjectivesPorphyromonas gingivalis (P.g.) is discussed to be involved in triggering self-reactive immune responses. The aim of this study was to investigate the autocitrullinated prokaryotic peptidylarginine deiminase (PPAD) from P.g. CH2007 (RACH2007-PPAD) from a rheumatoid arthritis (RA) patient and a synthetic citrullinated PPAD peptide (CPP) containing the main autocitrullination site as potential targets for antibody reactivity in RA and to analyse the possibility of citrullinating native human proteins by PPAD in the context of RA.MethodsRecombinant RACH2007-PPAD was cloned and expressed in Escherichia coli. Purified RACH2007-PPAD and its enzymatic activity was analysed using two-dimensional electrophoresis, mass spectrometry, immunoblot and ELISA. Autoantibody response to different modified proteins and peptides was recorded and bioinformatically evaluated.ResultsRACH2007-PPAD was capable to citrullinate major RA autoantigens, such as fibrinogen, vimentin, hnRNP-A2/B1, histone H1 and multiple peptides, which identify a common RG/RGG consensus motif. 33% of RA patients (n=30) revealed increased reactivity for α-cit-RACH2007-PPAD before RA onset. 77% of RA patients (n=99) presented α-cit-specific signals to CPP amino acids 57–71 which were positively correlated to α-CCP2 antibody levels. Interestingly, 48% of the α-CPP-positives were rheumatoidfactor IgM/anti-citrullinated peptide/protein antibodies (ACPA)-negative. Anti-CPP and α-RACH2007-PPAD antibody levels increase with age. Protein macroarrays that were citrullinated by RACH2007-PPAD and screened with RA patient sera (n=6) and controls (n=4) uncovered 16 RACH2007-PPAD citrullinated RA autoantigens and 9 autoantigens associated with lung diseases. We showed that the α-CPP response could be an important determinant in parenchymal changes in the lung at the time of RA diagnosis (n=106; p=0.018).ConclusionsRACH2007-PPAD induced internal citrullination of major RA autoantigens. Anti-RACH2007-PPAD correlates with ACPA levels and interstitial lung disease autoantigen reactivity, supporting an infection-based concept for induction of ACPAs via enzymatic mimicry.

Background There is a need for biomarker to identify patients “at risk” for rheumatoid arthritis (risk-RA) and to better predict the therapeutic response and in this study we tested the hypothesis that novel native and citrullinated heterogeneous nuclear ribonucleoprotein (hnRNP)-DL autoantibodies could be possible biomarkers. Methods Using protein macroarray and ELISA, epitope recognition against hnRNP-DL was analysed in sera from different developed RA disease and diagnosed SLE patients. Toll-like receptor (TLR) 7/9 and myeloid differentiation primary response gene 88 (MyD88)-dependency were studied in sera from murine disease models. HnRNP-DL expression in cultivated cells and synovial tissue was analysed by indirect immunofluorescence, immunoblot and immunohistochemistry. Results HnRNP-DL was highly expressed in stress granules, citrullinated in the rheumatoid joint and targeted by autoantibodies either as native or citrullinated proteins in patient subsets with different developed RA disease. Structural citrullination dependent epitopes (SCEs) of hnRNP-DL were detected in 58% of the SLE patients although 98% of these sera were α-CCP-2-negative. To obtain a specific citrullinated signal value, we subtracted the native antibody value from the citrullinated signal. The citrullinated/native index of autoantibodies against hnRNP-DL (CNDL-Index) was identified as a new value for an “individual window of treatment success” in early RA and for the detection of RF IgM/α-CCP-2 seronegative RA patients (24–46%). Negative CNDL-index was found in SLE patients, risk-RA and early RA cohorts such as EIRA where the majority of these patients are DAS28-responders to methotrexate (MTX) treatment (87%). High positive CNDL-values were associated with more severe RA, shared epitope and parenchymal changes in the lung. Specifically, native α-hnRNP-DL is TLR7/9-dependent, associated with pain and ROC analysis revealed an association to initial MTX or etanercept treatment response, especially in seronegative RA patients. Conclusion CNDL-index defines people at risk to develop RA and the “window of treatment success” thereby closing the sensitivity gap in RA.

BackgroundThere is a need for biomarker to identify patients ‘at risk’ for rheumatoid arthritis (risk-RA) and to better predict the therapeutic response and in this study we tested the hypothesis that novel native and citrullinated heterogeneous nuclear ribonucleoprotein (hnRNP)-DL autoantibodies could be possible biomarkers.MethodsUsing Protein macroarray and ELISA, epitope recognition against hnRNP-DL was analysed in sera from different developed RA disease and diagnosed SLE patients. Toll-like receptor (TLR) 7/9 and myeloid differentiation primary response gene 88 (MyD88)-dependency were studied in sera from murine disease models. HnRNP-DL expression in cultivated cells and synovial tissue was analysed by indirect immunofluorescence, immunoblot and immunohistochemistry. ResultshnRNP-DL was highly expressed in stress granules, citrullinated in the rheumatoid joint and targeted by autoantibodies either as native or citrullinated proteins in patient subsets with different developed RA disease. Structural citrullination dependent epitopes (SCEs) of hnRNP-DL were detected in 58% of the SLE patients although 98% of these sera were α-CCP2-negative. To obtain a specific citrullinated signal value, we subtracted the native antibody value from the citrullinated signal. This CNDL (Citrullinated-Native-hnRNP-DL)-index identified and the bioinformatic value was explored, as a new value for an “individual window of treatment success” in early RA and for the detection of RF-IgM/α-CCP2 seronegative RA patients (24-46%). Negative CNDL-index was found in SLE patients, risk-RA- and early RA-cohorts such as EIRA where the majority of these patients are DAS28-responders to methotrexate (MTX) treatment (87%). High positive CNDL-values were associated with more severe RA, shared epitope and parenchymal changes in the lung. Specifically, native α-hnRNP-DL is TLR7/9-dependent, associated with pain and ROC-analysis revealed an association to initial MTX or etanercept treatment response, especially in seronegative RA patients.ConclusionCNDL-index defines patients is a possible biomarker for develop RA and the “window of treatment success” thereby potentially closing the sensitivity gap.

BackgroundPrevious reports showed that peptidylarginine deiminase (PPAD) form Porphyromonas gingivalis (P.g.) is not able to citrullinate proteins internally. New mutated PPAD (mPPAD) from P.g. involved in periodontal disease (PD) cloned out of P.g. strain was characterised and analysed for its reactivity in sera from patients with systemic autoimmune diseases.MethodsRecombinant mutated PPAD from P.g. mPPAD mutations and citrullination sites were analysed by DNA sequencing and/or protein mass spectrometry. Autocitrullination activity it´s enzymatic-activity and human autoantigen protein citrullination was investigated by 2D-Elektrophoresis, MS, immunoblot analysis and ELISA. Furthermore we tested anti-mPPAD/cit-mPPAD with human sera (n=93) from early RA before and after onset of RA (n=30), established RA (n=32), SLE (n=16) and healthy blood donors (n=15) in ELISA assays. In RA mouse model collagen antibody-induced arthritis (CAIA), mPPAD-containing vesicles from P.g. were injected by intraperitoneal injection (IP).ResultsRecombinant mPPAD lacks 43 amino acids at the N-terminus and exhibits so far two new amino acid mutations (aminoacid position 73 (F>L) and 447 (E>V). We were able to demonstrate, mPPAD is enzymatically active over a huge pH-range (3-10) and autocitrullinates at amino acid position 63 the arginine to citrulline. Moreover mPPAd citrullinates major autoantigens in RA (Fibrinogen, Vimentin and hnRNP-A2/B1) which are detectable by RA patient sera and specific anti-citrulline monoclonal antibodies. mPPAD citrullinates HeLa-protein extracts and these specific citrullinated proteins are recognised by RA patient sera. Anti-citrullinated mPPAD antibodies were detected in 41% (n=32) of patients with RA but not in SLE (n=16) and control sera (n=15). In a RA follow-up study (n=30), we detected nearly similar antibody-sensitivities for citrullinated mPPAD before and after onset of RA (13/20%). Only a minority (7%) of RA patients show higher mPPAD antibody levels after RA diagnosis. In the CAIA RA mouse model mPPAD containing P.g. vesicles when injected IP showed a TLR2-dependent protective anti-inflammatory effect like P.g. LPS and Lipomannan.ConclusionsPg. infection and RA disease diagnosis occurs at different time points and Pg. infection induces a TLR2-dependent protective anti-inflammatory effect.We show the first time that mPPAD can citrullinate major human autoantigens internally and their immunologically and diagnostic relevance in RA.

PADs, through a hapten/carrier model. Here, we test this model in mice. Methods HLA-DRB1*04:01 transgenic mice were immunised subcutaneously with PADs or phosphate buffered saline (PBS) in Freund's complete adjuvant (CFA). Three booster injections of PAD or PBS in Freund' incomplete adjuvant (IFA) were given subcutaneously 15, 35 and 55 days later. Mice were: 1. tested for anti-PAD antibodies by ELISA. 2. tested for T cell responses to PADs, native or citrullinated fibrinogen 65 days after PAD immunisation. 3. tested for anti-citrullinated fibrinogen antibodies by ELISA using fibrinogen peptides under citrullinated and native form.Results HLA-DRB1*04:01 transgenic mice immunised with PADs developed antibodies and T cells to PADs and IgG antibodies to citrullinated peptides from fibrinogen, in the absence of T cell response to native or citrullinated fibrinogen. Conclusions T cell immunisation to PAD proteins triggers ACPAs through a hapten carrier mechanism in which the carrier is PAD which performs citrullination and the hapten any protein being citrullinated by PAD. Introduction Differences in enzymatic activity and pathogenic impact of Porphyromonas gingivalis (P.g.) peptidylarginine deiminase (PPAD) for development of RA have been published, confounded by different PPAD variations and methods used. Objectives Enzymatic active PPAD isolated from an RA patient (RA-PPAD) was first time linked to citrullination of RA autoantigens, diagnosis, therapy response and RA-onset. Methods Recombinant RA-PPAD cloned, verified by DNA sequencing and expressed in Escherichia coli and purified. RA-PPAD and its enzymatic activity was analysed using 2D-Elektrophoresis, mass spectrometry (MS), immunoblot and ELISA. Results RA-PPAD autocitrullinates amino acid position 63 (aa 63 ) and exhibits so far two new amino acid mutations aa 73 F to L and aa 447 E to V. Anti-citrullinated RA-PPAD antibodies were detected in 38% (n=36) of patients with RA, but were absent in Systemic lupus erythematosus (n=30), Osteoarthritis (n=36) and control sera (n=23). Twenty percent of RA patients (n=30) showed an increase in antibody-titre against citrullinated RA-PPAD after RA onset. High antibody titre against the cit-PPAD-peptide of 15aa (CPP) derived from the autocitrullination site (R 63 ) correlates with TNFa-inhibitor (TBA) non-response (n=61). Anti-CPP levels correlate with DAS28,rheumatoid factor, a-CCP 2 levels and increase with age. RA-PPAD is able to citrullinate internal arginines in fibrinogen, vimentin, hnRNP-A2/B1 and histone H1. This internal citrullination-sites are recognised by RA sera and able to bind HLA401. Conclusions Failure of P.g. clearance in RA patients may lead to excessive exposure of citrullinated self-antigens and bacterial antigens inducing immune-mimicry. P.g. infection can be linked to RA and its correlation to TBA non-response leads to the suggestion to clear P.g infection before a-TNF treatment. Introduction Genome-wide association studies of multiple autoimmune diseases, including Sjögren's syndrome, systemic lu...

ObjectivesPrevious reports showed that peptidylarginine deiminase (PPAD) form Porphyromonas gingivalis (P.g.) is not able to citrullinate proteins internaly. New mutated PPAD (mPPAD) from P.g. involved in periodontal disease (PD) cloned out of P.g. strain was characterized and analyzed for its reactivity in sera from patients with systemic autoimmune diseasesMethodsWe cloned a new enzymatically active recombinant mutated PPAD from P.g. mPPAD mutations and citrullination sites were analyzed by DNA sequencing and/or protein mass spectrometry. Autocitrullination activity it's enzymatic-activity and human autoantigen protein citrullination was investigated by 2D-Elektrophoresis, MS, immunoblot analysis and ELISA. Furthermore we tested anti-mPPAD/cit-mPPAD with human sera (n=93) from early RA before and after onset of RA (n=30), established RA (n=32), SLE (n=16) and healthy blood donors (n=15) in ELISA assays. To study a potential impact on the RA mouse model (CAIA), mPPAD-containing vesicles from P.g. were injected by intraperitoneal injection (IP).ResultsRecombinant mPPAD lacks 43 amino acids at the N-terminus and exhibits so far two new amino acid mutations (amino acid position 73 (F>L) and 447 (E>V). We were able to demonstrate, mPPAD is enzymatically active over a huge pH-range (3–10) and autocitrullinates at amino acid position 63 the arginine to citrulline. Moreover mPPAd citrullinates major autoantigens in RA (Fibrinogen, Vimentin and hnRNP-A2/B1) which are detectable by RA patient sera and specific anti-citrulline monoclonal antibodies. mPPAD citrullinates HeLa-protein extracts and these specific citrullinated proteins are recognized by RA patient sera. Anti-citrullinated mPPAD antibodies were detected in 41% (n=32) of patients with RA but not in SLE (n=16) and control sera (n=15). In a RA follow-up study (n=30), we detected nearly similar antibody-sensitivities for citrullinated mPPAD before and after onset of RA (13/20%). Only a minority (7%) of RA patients show higher mPPAD antibody levels after RA diagnosis. In the Collagen antibody-induced arthritis (CAIA) RA mouse model mPPAD containing P.g. vesicles when injected IP showed a TLR2-dependent protective anti- inflammatory effect like P.g. LPS and Lipomannan.ConclusionsPg. infection and RA disease diagnosis occurs on different timepoints and Pg. infection induces a TLR2-dependent protective anti-inflammatory effect.We show the first time that mPPAD can citrullinate major human autoantigens internally and their immunologically and diagnostic relevance.Disclosure of InterestNone declared

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ObjectiveMonoclonal Anti-citrullinated protein autoantibodies ACPA target different proteins and their role in RA is still largely unknown. We analysed the susceptibility of PWD Musmusculusmusculus subspecies, separated from Musmusculusdomesticus about one million years ago to collagen antibody-induced arthritis (CAIA) and the role of ACPA antibodies in the CAIA model of arthritis.MethodsCAIA was induced by administration of collagen antibodies followed by lipopolysaccharide injection. Two genetically distinct mouse strains, representatives of the subspecies Mus musculus domesticus and Mus musculus musculus were analysed for the development of clinical and histological signs of arthritis upon CAIA treatment. ACPA antibodies were tested by adding it to the CAIA cocktail. Autoantigenic profile was generated using protein macroarrays.ResultsThe wild-derived mouse strain PWD/Ph was highly susceptible to CAIA induced arthritis, whereas the classical laboratory strain C57BL/6J was resistant. Mice carrying chromosomes 5 or 12 from PWD on a B6 background display a B6-like phenotype in the CAIA model as well as the F1 hybrids (B6xPWD and PWDxB6) implicating the presence of dominant resistance modifiers in the C57BL/6J genetic background. The two mouse strains differ highly in their autoantigenic profile. Injecting specific monoclonal ACPAs reactive with the citrullintated H1 and H4 were able to block the CAIA induced arthritis. This inhibition can be explained in part by a Toll 9 dependent inhibition of osteoclasts. Moreover TLR2 activation via P.gingivalis LPS and lipomannan treated animals show a 80% reduction of arthritis score compared to E. coli LPS in a C57BL/6J CAIA model. Anti-collagen specific antibodies are increased in both strains B6 and PWD when arthritis is induced. Commercial CCP2 ELISA detected ACPA in animals regardless of the treatment. Using the arginine and citrulline containing peptides as control shows that this response is directed to the arginine containing peptide.ConclusionsThe Mus musculus musculus derived mouse strain PWD/Ph is highly susceptible to arthritis development in the CAIA model. TLR2 activation blocks CAIA and specific ACPAs are able to block Osteoclast activation via TLR 9 activation and these monoclonal antibodies may be used as therapeutic antibodies in the future that protect from RA development.