Background and Objectives Rheumatoid arthritis (RA) is characterised by the presence of anti-citrullinated peptide antibodies (ACPA) years before disease onset. Increasing molecular and epidemiological evidence has linked periodontitis (PD) to RA. Porphyromonas gingivalis is unique amongst periodontal pathogens in possessing a citrullinating enzyme, peptidylarginine deiminase (PPAD) with the potential to generate citrullinated antigens driving the autoimmune response in RA. We have examined the immune response to several peptides/proteins of significance to RA in DBA/1 mice immunised with recombinant PPAD.
Materials and Methods Twelve week old DBA/1 mice were immunised with one of two emulsions: 1) recombinant PPAD in complete Freund’s adjuvant (CFA) or 2) an inactive PPAD mutant (C351A) in CFA. Clinical score and paw swelling of mice (indicative of arthritis) recorded for ten days post onset. Antibody responses to PPAD and C351A, and a number of immunodominant ACPA target peptides: anti-citrullinated a-enolase peptide-1 (CEP1), vimentin (cVim), fibrinogen (cFib) and their uncitrullinated forms (REP-1, vim and fib) were examined in mouse serum using Enzyme-linked Immunosorbant assays (ELISAs). The Mann-Whitney U test was used to calculate p-values for differences between the sera groups for each antigen.
Results By day 30 post immunisation, 20% of mice immunised with PPAD had developed arthritis-like swelling in their paws. There was no significant difference between the antibody response to PPAD and the antibody response to C351A in any of the mice tested. There was a significantly raised antibody response (p < 0.05) to both CEP1 and REP1 (mean 0.263; OD450) in the mice immunised with PPAD compared to the mice immunised with C351A (CEP1, mean 0.074 (OD450) and REP1 mean 0.150 (OD450). Antibody responses to cFib and Fib were similar in all mice, as were antibody responses to cVim and Vim.
Conclusions The paw swelling and raised immune response to the immunodominant enolase peptide, both citrullinated (CEP1) and uncitrullinated (REP1), in mice immunised with autocitrullinated PPAD shows that PPAD induces arthritis and autoimmunity to enolase. This demonstrates that an active citrullinating PPAD can break tolerance to a major RA autoantigen and provides further molecular evidence linking P. gingivalis infection to RA.