Bacterial citrullinated epitopes generated by Porphyromonas gingivalis infection-a missing link for ACPA production

Abstract

Objectives: Porphyromonas 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 (RA_CH2007-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.

Methods: Recombinant RA_CH2007-PPAD was cloned and expressed in Escherichia coli. Purified RA_CH2007-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.

Results: RA_CH2007-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-RA_CH2007-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-RA_CH2007-PPAD antibody levels increase with age. Protein macroarrays that were citrullinated by RA_CH2007-PPAD and screened with RA patient sera (n=6) and controls (n=4) uncovered 16 RA_CH2007-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).

Conclusions: RA_CH2007-PPAD induced internal citrullination of major RA autoantigens. Anti-RA_CH2007-PPAD correlates with ACPA levels and interstitial lung disease autoantigen reactivity, supporting an infection-based concept for induction of ACPAs via enzymatic mimicry.

Keywords: autoantibodies; autoimmunity; early rheumatoid arthritis; infections.