Peptidylarginine deiminase 2 is required for tumor necrosis factor alpha-induced citrullination and arthritis, but not neutrophil extracellular trap formation

Abstract

Citrullination, the post-translational conversion of arginines to citrullines, may contribute to rheumatoid arthritis development given the generation of anti-citrullinated protein antibodies (ACPAs). However, it is not known which peptidylarginine deiminase (PAD) catalyzes the citrullination seen in inflammation. PAD4 exacerbates inflammatory arthritis and is critical for neutrophil extracellular traps (NETs). NETs display citrullinated antigens targeted by ACPAs and thus may be a source of citrullinated protein. However, PAD4 is not required for citrullination in inflamed lungs. PAD2 is important for citrullination in healthy tissues and is present in NETs, but its role in citrullination in the inflamed joint, NETosis and inflammatory arthritis is unknown. Here we use mice with TNFα-induced inflammatory arthritis, a model of rheumatoid arthritis, to identify the roles of PAD2 and PAD4 in citrullination, NETosis, and arthritis. In mice with TNFα-induced arthritis, citrullination in the inflamed ankle was increased as determined by western blot. This increase was unchanged in the ankles of mice that lack PAD4. In contrast, citrullination was nearly absent in the ankles of PAD2-deficient mice. Interestingly, PAD2 was not required for NET formation as assessed by immunofluorescence or for killing of Candida albicans as determined by viability assay. Finally, plasma cell numbers as assessed by flow cytometry, IgG levels quantified by ELISA, and inflammatory arthritis as determined by clinical and pathological scoring were all reduced in the absence of PAD2. Thus, PAD2 contributes to TNFα-induced citrullination and arthritis, but is not required for NETosis. In contrast, PAD4, which is critical for NETosis, is dispensable for generalized citrullination supporting the possibility that NETs may not be a major source of citrullinated protein in arthritis.

Mechanisms and disease relevance of neutrophil extracellular trap formation

“…In fact, under such conditions where NETs may not contribute to antimicrobial action per se, excessive NET formation and the release of neutrophil intracellular DAMPs may cause unchecked inflammation and tissue damage, thereby affecting host survival. Indeed, the absence of NET formation in PAD4‐deficient mice protects these animals from LPS‐induced shock, suggesting that NETs contribute to the unrestrained inflammatory and procoagulant host response to LPS in this model system . Mice lacking PAD4 did not show enhanced susceptibility to infection with influenza virus, but DNase treatment and depletion of neutrophils rescued mortality in models of severe flu, and NETs have been associated with increased inflammation in pulmonary infections with Sendai virus .…”

“…Indeed, the absence of NET formation in PAD4-deficient mice protects these animals from LPS-induced shock, suggesting that NETs contribute to the unrestrained inflammatory and procoagulant host response to LPS in this model system. 7,35 Mice lacking PAD4 did not show enhanced susceptibility to infection with influenza virus, 37 but DNase treatment and depletion of neutrophils rescued mortality in models of severe flu, 38 and NETs have been associated with increased inflammation in pulmonary infections with Sendai virus. 39 Viewing these and other previous studies in combination, current concepts propose that NET release drives and maintains excessive inflammation, autoimmunity and tissue damage, rather than augmenting host defence, [40][41][42] an issue requiring further in vivo studies.…”

“…Another histone modification involved in NETosis is histone citrullination, which is driven by the nuclear enzyme protein‐arginine deiminase 4 (PAD4) . PAD4‐deficient neutrophils fail to form NETs in response to lipopolysaccharide (LPS) and tumour necrosis factor (TNF) . Decondensed chromatin is released into the cytoplasm of intact cells and binds NE and other granular enzymes and toxins (Figure ).…”

“…Specifically, NET‐associated histones are citrullinated and exposed to circulating antibodies, providing a potential source of autoantigens and autoantibody production. Despite these findings, murine RA models using PAD4 (enzyme driving protein citrullination in neutrophils) knock‐out mice to interrogate NET contribution in vivo, revealed discrepant results depending on the arthritis model used and relative contributions of different PADs in the microenvironmental RA context . Among dermatologic indications, neutrophilic inflammation and NETosis have attracted particular attention for psoriasis where neutrophils are found as major infiltrating immune cell in skin histologies, yet their functional relevance in disease initiation or progression remains largely elusive.…”

“…6 PAD4-deficient neutrophils fail to form NETs in response to lipopolysaccharide (LPS) and tumour necrosis factor (TNF). 7 Decondensed chromatin is released into the cytoplasm of intact cells and binds NE and other granular enzymes and toxins ( Figure 1).…”