Peptidylarginine deiminase 4 contributes to tumor necrosis factor α-induced inflammatory arthritis.

Rheumatoid arthritis has long been known to be an inflammatory arthritis, with tumor necrosis factor α (TNFα) playing a leading role. However, more recent evidence demonstrates that rheumatoid arthritis is also an autoimmune disease characterized by autoantibodies such as anti–citrullinated protein antibodies (ACPAs). ACPAs are specific for rheumatoid arthritis, predictive of more severe disease, and implicated in disease pathogenesis (1). Although the pathophysiology of rheumatoid arthritis is incompletely understood, it is hypothesized (2,3) that rheumatoid arthritis can be triggered by protein citrullination, potentially from environmental exposures such as tobacco smoke (4) or Porphyromonas gingivalis infection (5), followed by the development of ACPAs in genetically predisposed individuals. The incorporation of citrullinated antigens into ACPA immune complexes can result in immune complex deposition in the joint stimulating macrophage activation, TNFα production, inflammation, and ultimately clinical rheumatoid arthritis. However, many of the factors that lead to protein citrullination, ACPAs, and arthritis are not clearly defined. Citrullination is the conversion of a protein’s arginine residues to citrulline, and it is catalyzed by peptidylarginine deiminases (PADs). Citrullination is increased in the rheumatoid joint (6), and inhibition of PADs with Cl-amidine decreases murine collagen-induced arthritis (7), supporting a role for the PADs in rheumatoid arthritis. Since PAD2 and PAD4 are expressed in inflammatory cells and up-regulated in inflamed joints (8), they may be the main PADs responsible for citrullination in arthritis. PAD4 is particularly interesting since its gene contains single-nucleotide polymorphisms associated with rheumatoid arthritis (9). Further, PAD4 is critical for the formation of neutrophil extracellular traps (NETs) (10), which are inflammatory and present some of the same citrullinated antigens that can be targeted by ACPAs (11). Thus, PAD4 could contribute to rheumatoid arthritis pathogenesis due to a role in inflammation and/or antigen citrullination. However, PAD4 is not essential for acute murine K/BxN arthritis (12), a model of the effector component of rheumatoid arthritis that is dependent upon neutrophils (13). Thus, the role of PAD4 in rheumatoid arthritis remains unclear. Fully understanding the contributions of PAD4 to rheumatoid arthritis is particularly important, since drugs targeting PAD4 are under development (14). As mentioned above, protein citrullination is sometimes considered a starting point for the development of rheumatoid arthritis (2), but citrullination is associated with inflammation of many types (15) and may be a consequence of rheumatoid inflammation (16) as well as a trigger. Interestingly, TNFα, which is present at high levels in rheumatoid arthritis, can induce PAD4 nuclear translocation, histone citrullination (17,18), and NET formation (11,19). Therefore, TNFα may propagate inflammation in rheumatoid arthritis in part through PAD4. Further, TNFα is known to positively regulate B cell proliferation and antibody production (20,21) and could thus augment ACPA production. Indeed, the ACPA repertoire expands and TNFα levels increase prior to the development of clinical rheumatoid arthritis (22), but it has been hypothesized that TNFα up-regulation is downstream of antigen citrullination and ACPA production (2). This idea is consistent with the ability of citrullinated proteins and ACPAs to induce TNFα production by macrophages (23). However, the ability of ACPA immune complexes to induce TNFα does not exclude the possibility that TNFα could also augment ACPA production. There could be a complex positive feedback network involving TNFα, PAD4, citrullination, and ACPAs driving rheumatoid arthritis, but most work has focused on citrullination and autoantibodies upstream of TNFα, not downstream. Overexpression of TNFα in mice causes a chronic, erosive inflammatory arthritis similar to rheumatoid arthritis (24), but little is known about the production of autoantibodies or the role of PAD4 in this model. Here we show that overexpression of TNFα amplifies autoantibody production, and PAD4 mediates TNFα-induced autoantibodies, inflammation, and chronic inflammatory arthritis.