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Objective. First-degree relatives (FDRs) of rheumatoid arthritis (RA) patients sharing genetic and environmental risk factors for RA may represent a pre-RA state. Since anti-cyclic citrullinated protein/ peptide antibodies (ACPAs) appear years before the onset of RA, the purpose of this study was to determine the prevalence of various ACPAs in FDRs of RA patients.Methods. We evaluated 88 RA patients, 50 unaffected FDRs, and 20 healthy control subjects. Conclusion. The rate of ACPA positivity in unaffected FDRs of RA patients with a high prevalence of the SE and smoking was 48%, whereas ACPAs were rare in the healthy controls. ACPAs in the FDRs of RA patients was most commonly of the IgA isotype, but IgG ACPA targeting citrullinated vimentin was also frequently found.

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Rheumatoid Arthritis (RA) is more common and severe in women compared to men. Both women and men with RA express autoantibodies to post-translationally modified antigens, including citrullinated and homocitrullinated proteins or peptides. These autoantibodies are strongly linked with the HLA-DR4 gene. The objective of this study was to determine sex differences in immune responses to homocitrullinated antigens. We used a humanized animal model of RA, DR4-transgenic mice and immunized them with a homocitrullinated peptide called HomoCitJED. Immune responses in these mice were measured for splenocyte proliferation by tritiated thymidine incorporation, serum autoantibody production by ELISA and cytokine levels by multiplex. We found that T cell and antibody responses to homocitrullinated antigens were similar in male and female mice. However, we found sex differences in serum cytokine profiles with female mice having higher ratio of IL-1α to IL-5, suggesting imbalances in immune regulation. This is the first study to report that immune responses to homocitrullinated antigens can be differentiated by sex.

IntroductionPeriodontitis (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 methodsCultured 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.ResultsPgEnolase, 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.ConclusionPPAD, 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.