Background/Purpose: Antibodies are a cornerstone of immunity and pathologic in autoimmunity. Immunoglobulin G (IgG) can undergo multiple different post-translation modifications, some of which can alter IgG half-life, but citrullination has not been evaluated in this context. Citrullination, the post-translational conversion of arginines to citrullines, as well as the citrullinating enzymes peptidylarginine deiminase (PAD) 2 and 4, are increased in inflammation and rheumatoid arthritis. Further, citrullinated IgG has been detected in human rheumatoid arthritis and both PAD2 and PAD4 are required for normal IgG levels in a murine model of rheumatoid arthritis. However, the role of these PADs in a normal antibody response and IgG half-life is unknown. The purpose of this study is to determine if PAD2 or PAD4 is required for a normal IgG response to a T cell dependent immunization and normal IgG half-life.

Methods: Wild-type mice, PAD2-/- mice, PAD4-/- mice, wild-type mice treated daily with the pan-PAD inhibitor Cl-amidine, and wild-type mice treated with vehicle control were immunized and then boosted at day 42 with 4-hydroxy-3-nitrophenylacetyl conjugated to keyhole limpet hemocyanin (NP-KLH) precipitated in alum to generate a T cell dependent immune response against NP. Serum from multiple time points was subjected to enzyme-linked immunosorbent assay (ELISA) to detect anti-NP IgG. To assess IgG half-life, serum from NP-KLH immunized mice was transferred to naïve mice. Serum was collected from recipients and the loss of NP-specific IgG quantified by ELISA over time. Similar studies were performed with transferred purified IgG from NP-KLH immunized PAD2-/- and PAD2+/+ mice after in vitro citrullination with PAD2. The Fc portion of IgG was purified and citrullination assessed by anti-modified citrulline (AMC) western blot.

Results: PAD2-/-, PAD4-/-, and Cl-amidine treated wild-type mice have reduced IgG titers in response to NP-KLH compared to untreated or vehicle control-treated wild-type mice. NP-specific IgG titers fall faster in recipients of serum from PAD2-/-, PAD4-/-, and Cl-amidine treated mice than recipients of serum from untreated or vehicle control-treated wild-type mice, with a more pronounced effect with serum from PAD2-/- donors compared to wild-type than PAD4-/- donors compared to wild-type. IgG is citrullinated in healthy mice and citrullination of the Fc portion of IgG is reduced as detected by AMC blot in PAD2-/-, but not PAD4-/- mice. In vitro citrullination rescues the half-life of IgG from PAD2-/- mice.

Conclusion: IgG is citrullinated and PAD enzymes, especially PAD2, are required for normal IgG levels in response to immunization as well as normal IgG half-life in mice. These findings have implications for immunity, for monoclonal antibody based therapies in rheumatoid arthritis, and for both the longevity and specificity of rheumatoid autoimmune antibodies, which bind to citrullinated proteins and IgG.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/peptidylarginine-deiminases-are-required-for-normal-immunoglobulin-g-half-life/