Background/Purpose: There is increasing molecular and epidemiological evidence linking Porphyromonas gingivalis to RA. P. 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. To examine the effect of PPAD on collagen induced arthrititis (CIA), we co-immunised C57BL/6 mice with PPAD and collagen and measured disease outcome by clinical scores, histology and antibody responses. 

Methods: Male C57BL/6 animals (8-10 weeks, n=15/group) were immunised with 200ug type II collagen (cII) in CFA alone, or cII emulsified in 50ug PPAD or emulsified in 50ug of an enzymatically inactive PPAD mutant control (C531A) in CFA at the base of the tail.  Mice were monitored daily for the first appearance of peripheral joint oedema.  Clinical scores and paw swelling measurements were recorded daily and mice were culled after 10 days of disease onset.  Their joints were fixed in 10% buffered formalin and decalcified with 10%EDTA for histological analysis, and serum was collected by terminal cardiac puncture for antibody titres. Antibodies to collagen, CCP2 and recombinant PPAD were measured by ELISA.

Results: Between days 5-10 post disease onset, PPAD immunised mice had a significantly increased clinical score of arthritis (mean = 6.83, p=0.0126) compared to cII alone immunised mice (mean= 3.40).  There was a minor increase in the clinical scores when C351A immunised mice (mean=4.77) were compared to mice immunised with cII alone, but this did not reach statistical significance (p=0.290). This correlated with an increase in PPAD-specific antibodies (p< 0.0001) in PPAD immunised mice when compared to the mutant C531A or cII immunised mice.  There was no increase in anti-collagen antibodies in the PPAD immunised mice and anti-CCP2 antibodies where negative in all 3 groups. Histological analysis showed that PPAD immunisation resulted in increased synovitis and joint erosion (p=0.021) when compared to cII controls.    

Conclusion: PPAD immunisation exacerbated both clinical and histological parameters of CIA.  There was no significant difference between the mice immunised with cII alone and the mice co-immunised with the enzymatically inactive mutant (C351A), supporting the hypothesis that enzymatic activity of PPAD is necessary to drive its arthritogenic effect. These data indicate a molecular mechanism that explains the link between periodontitis and RA and suggests potential therapeutic benefit by targeting PPAD.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/porphyromonas-gingivalis-peptidylarginine-deiminase-exacerbates-collagen-induced-arthritis-in-c57bl6-mice/