Background/Purpose: Agonistic antibodies to Peptidylarginine Deiminase 4 (PAD4) are hallmarks of a severe form of rheumatoid arthritis (RA) characterized by the most erosive joint damage and lung disease. PAD4 activation and the production of citrullinated autoantigens targeted in RA is likely a major mechanism by which these antibodies may worsen the disease. However, whether anti-PAD4 antibodies may have other effector functions in RA is unknown. The recent discovery that PAD4 is found on the surface of monocytes drove our interest to define the consequences that anti-PAD4 binding may have on the function of these cells.

Methods: We investigated the consequences of PAD4-activating antibody binding to monocytes using a well-characterized panel of human anti-PAD4 and control monoclonal antibodies (mAbs). Extracellular citrullination of fibrinogen was used to study antibody-mediated PAD4 activation on surface of monocytes. We measured anti-PAD4 antibody surface binding and monocyte activation using flow cytometry, and release of pro-inflammatory cytokines using ELISA at 24 hours. We assessed differentiation of monocytes into osteoclasts using tartrate resistant acid phosphatase (TRAP) staining and quantitative estimation of acid phosphatase (AP) activity in the presence of a sub-optimal dose of RANKL at 14 days.

Results: PAD4 is expressed on the monocyte surface in an enzymatically active state that can be hyperactivated twofold upon binding by PAD4 antibodies. Flow cytometry studies revealed preferentially binding of PAD4 mAbs to PAD4 present on surface of monocytes where 63% monocytes bound with PAD4 mAbs versus 1.9% binding with control mAbs (p= 0.0087). Moreover, PAD4 mAbs but not control mAbs induced monocyte activation with upregulation of surface markers such as CD40, CD11c and PDL-1 after 24 hours. Other activation markers such as CD80, CD86 and HLA-DR remained unchanged. Interestingly, monocytes treated with PAD4 mAbs released 100-fold more RA-associated pro-inflammatory cytokines, including IL-6 (1440 pg/ml) and TNF-alpha (94pg/ml), compare to monocytes incubated with control mAbs (14 pg/ml and 0 pg/ml, respectively). At 14 days, we observed larger osteoclast-like cells that were positively stained for TRAP in the presence of PAD4 mAbs compared to control mAbs. This corresponded to a three-fold increase in the amount of AP released by cells cultured with PAD4 mAbs over control mAbs.

Conclusion: Anti-PAD4 autoantibodies can directly interact with monocytes and influence their pathophysiological functions. The pro-inflammatory activation and differentiation into osteoclast-like cells may contribute to the erosive joint disease observed in this severe RA subset.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/antibodies-to-pad4-drive-monocyte-activation-and-differentiation-into-osteoclast-like-cells/