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.