Macrophage extracellular traps require peptidylarginine deiminase 2 and 4 and are a source of citrullinated antigens bound by rheumatoid arthritis autoantibodies

Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis, but the sources of citrullinated antigens remain incompletely defined. Neutrophil extracellular traps (NETs) appear to be a source of ACPA targets. However, while the citrullinating enzyme peptidylarginine deiminase (PAD)4 is required for producing citrullinated NETs, it is not required for citrullination in murine arthritis. Conversely, PAD2 has a limited role in producing NETs but is a major contributor to citrullinated proteins. Thus, additional sources of citrullinated antigens likely exist. We investigated if macrophage extracellular traps (METs) could be a source of citrullinated proteins in a PAD2 or PAD4 dependent manner. Thioglycolate induced macrophages from wild-type, PAD2 −/−and PAD4 −/−mice were activated with a variety of stimulants, then fixed and stained with DAPI and either anti-citrullinated histone H4 (H4C3) antibody or sera from ACPA+ or ACPA− rheumatoid arthritis subjects. METs were visualized by immunofluorescence, confirmed to be extracellular using DNase, counted in a blinded manner, and compared by paired-T test. We found that ionomycin and monosodium urate (MSU) crystals induce citrullinated METs (H4C3 stained). In the absence of PAD2 or PAD4, citrullinated METs were reduced in response to ionomycin and MSU when compared to wildtype, with a complete absence of citrullinated METs in PAD4 −/−macrophages. Also, IgG from ACPA+ sera, but not ACPA− sera, bound to METs. Finally, in the absence of PAD2 or PAD4, ACPA bound METs were reduced in response to ionomycin and MSU. Thus, METs appear to contribute to the generation of citrullinated antigens targeted by ACPAs in rheumatoid arthritis in a PAD2− and PAD4-dependent manner. Funding for this work was provided by the U.S. Army MedicalResearch Acquisition Activity through the Peer Reviewed MedicalResearch Program (W81XWH-18-1-0717) to M.A.S.