Background/Purpose: Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis, but the sources of citrullinated antigens as well as which peptidylarginine deiminases (PADs) are required for their production remain incompletely defined. Neutrophil extracellular traps (NETs) appear to be a source of citrullinated antigens bound by ACPAs, but they may not be the only source. Macrophages also generate extracellular traps (METs), but their role in rheumatoid arthritis is still emerging. The purpose of this study was to determine if METs could be a source of citrullinated proteins bound by ACPAs, and if their formation requires PAD2 or PAD4.

Methods: Thioglycolate-elicited peritoneal macrophages were isolated from wildtype, PAD2^-/-, and PAD4^-/- mice and were left untreated or were treated with ionomycin, monosodium urate (MSU) crystals, platelet activation factor, tumor necrosis factor alpha, lipopolysaccharide, phorbol myristate acetate, or heat killed Candida albicans. Macrophages were fixed and incubated with DAPI and either anti-citrullinated histone H4 antibody or sera from five ACPA+ or ACPA- rheumatoid arthritis subjects. Five predetermined areas of each coverslip were visualized by immunofluorescence at 400x, and METs (decondensed structures determined to be extracellular by DNase degradation studies) were counted by eye in a blinded manner. The percentage of macrophages that were METs, citrullinated METs, and ACPA-bound METs were compared between wildtype and PAD-deficient macrophages by paired t-test with p< 0.05 considered significant.

Results: Ionomycin and MSU crystals reliably induced METs with citrullinated histones. In the absence of PAD2 or PAD4, citrullinated METs were reduced, with a complete absence of citrullinated METs in PAD4-deficient macrophages. Also, IgG from ACPA+, but not ACPA- sera, bound METs. Finally, in the absence of PAD2 or PAD4, ACPA-bound METs were lost.

Conclusion: In response to ionomycin and MSU, macrophages generate citrullinated extracellular traps in a PAD4- and PAD2-dependent manner.Furthermore, ACPA+ sera can bind METs, suggesting that METs may contribute to the generation of citrullinated antigens targeted by ACPAs in rheumatoid arthritis in a PAD2- and PAD4-dependent manner.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/macrophage-extracellular-traps-require-peptidylarginine-deiminase-2-and-4-and-are-a-source-of-citrullinated-antigens-bound-by-rheumatoid-arthritis-autoantibodies/