Background/Purpose: Most rheumatoid arthritis (RA) patients express autoantibodies to post-translationally modified antigens, including citrullinated and homocitrullinated (also known as carbamylated) peptides. Homocitrulline and citrulline are structurally similar and immune responses to peptides containing either of these modifications are cross-reactive. Citrullinated peptides bind strongly to MHCII molecules containing a consensus sequence called the Shared Epitope (encoded by HLA-DRB1 alleles, the strongest genetic risk factor for RA). The relationship between homocitrullinated peptides and the Shared Epitope remains unclear. The aim of this study is to determine whether homocitrullinated peptides induce arthritis in a humanized mouse model of RA expressing the Shared Epitope.

Methods: Male and female HLA-DRB1*04:01-transgenic (DR4tg) mice and wild-type C57Bl/6 (B6) mice were immunized with a synthetic homocitrullinated peptide (HomoCitJED) or PBS and then received intra-articular (i.a.) HomoCitJED in one knee and PBS in the other knee. Immune responses in these mice were measured serially for serum autoantibody production by ELISA to the following antigens: HomoCitJED, CitJED (a synthetic citrullinated peptide that contains the same backbone as HomoCitJED), homocitrullinated fibrinogen (HomoCitFib), citrullinated fibrinogen (CitFib), fibrinogen (Fib) and cyclic citrullinated protein/peptide 2 (CCP2).Arthritis was assessed by serial measurements of joint swelling with calipers and by histopathology at sacrifice (day 137 post-primary immunization) using a standardized scoring system.

Results: After immunization with HomoCitJED, all DR4tg mice (14/14) developed IgG anti-HomoCitJED and anti-HomoCitFib antibodies, compared to 4/6 and 3/6 B6 mice, respectively. A small number of mice developed antibodies to CitJED (4/14 DR4tg and 1/6 B6). Anti-CCP2 was detected in 8/14 DR4tg and 4/6 B6 mice. None of the mice had detectable antibodies to Fib or CitFib. Antibody levels did not change significantly after i.a. injections and over time. PBS immunized mice did not have detectable immune responses or arthritis. After day 78 post-immunization, significant joint swelling was observed in the knees of HomoCitJED injected DR4tg mice with an arthritis score of 3.92 (SD 2.90)/12 versus 1.18 (SD 1.08)/12 in HomoCitJED-injected B6 wild-type mice; p=0.0016. Histopathologic findings included synovial thickening and pannus, hypervascularity, cartilage destruction and bone erosions.

Conclusion: Mice expressing human Shared Epitope developed immune responses to homocitrullinated and citrullinated antigens and RA-like arthritis. These findings support an arthritogenic role for homocitrullinated antigens that is enhanced by the Shared Epitope.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/arthritogenicity-of-homocitrullinated-peptides-in-hla-drb1-transgenic-mice/