The monocyte cell surface as a novel site of autoantigen generation in Rheumatoid Arthritis

Abstract Citrullination is recognized as a key pathogenic process in rheumatoid arthritis (RA), as evidenced by the formation of anti-citrullinated protein antibodies (APCAs) in the majority of patients; however, the mechanisms that result in citrullinated autoantigen generation are not fully understood. Although the citrullinating enzyme peptidylarginine deiminase IV (PAD4) is predominantly expressed by neutrophils and monocytes, the contribution of monocytes to the citrullinated autoantigen pool has been underexplored. In this study, we utilized multiple complementary methods including flow cytometry, immunofluorescence, and transmission electron microscopy, which revealed a predominantly extranuclear localization of PAD4 in monocytes with a fraction present on the cell surface. Surface PAD4 was enzymatically active and citrullinated both extracellular fibrinogen and endogenous surface proteins in a calcium dose–dependent manner. In addition, human monoclonal ACPAs cloned from patients with RA recognized fibrinogen citrullinated by monocyte-surface PAD4. Mass spectrometry analysis of citrullinated proteins from the cell surface fraction revealed CD11b to be a novel PAD4 substrate. Citrullinated CD11b was recognized by autoantibodies in 60% of ACPA+ RA patients compared to 6% of healthy controls (p=0.0021) and 0% of ACPA− RA patients (p≤0.001). Taken together, our study demonstrates that PAD4 is expressed on the surface of monocytes in an enzymatically active state that renders the monocyte surface a novel site of citrullinated autoantigen generation in RA. Supported by funds received from Bristol-Myers Squibb.