Tumor necrosis factor alpha, citrullination, and peptidylarginine deiminase 4 in lung and joint inflammation

BACKGROUND: The relationship between lung and joint inflammation in rheumatoid arthritis is poorly understood. Lung inflammation with resultant protein citrullination may trigger anti-citrullinated protein antibodies, inflammation, and arthritis. Alternatively, lung and joint inflammation may be two manifestations of a single underlying pathology. The lung has increased citrullination and TNF-alpha levels are high in rheumatoid arthritis; however, it is unknown if TNF-alpha can induce lung protein citrullination. The citrullinating enzyme peptidylarginine deiminase 4 (PAD4) exacerbates TNF-alpha-induced arthritis, but a role for PAD4 in lung citrullination and TNF-alpha-induced lung inflammation has not been explored. Our aim was to use TNF-alpha-overexpressing mice to clarify the intersection of TNF-alpha, citrullination, PAD4, arthritis, and lung inflammation. METHODS: Lung protein citrullination in wild-type mice, mice that overexpress TNF-alpha systemically (TNF(+)), TNF(+)PAD4(+/+), and TNF(+)PAD4(-/-) mice was quantified by both gel electrophoresis using a citrulline probe and western blot. Hematoxylin and eosin (HandE)-stained lung sections from TNF(+)PAD4(+/+) and TNF(+)PAD4(-/-) mice were scored for lung inflammation. HandE-stained ankle joint sections from mice that overexpress TNF-alpha only in the lungs were assessed for arthritis. RESULTS: TNF(+) mice have increased lung protein citrullination. TNF(+)PAD4(-/-) mice do not have significantly reduced lung protein citrullination, but do have decreased lung inflammation compared to TNF(+)PAD4(+/+) mice. Mice that overexpress TNF-alpha only in the lungs do not develop arthritis. CONCLUSIONS: PAD4 exacerbates lung inflammation downstream of TNF-alpha without having a major role in generalized protein citrullination in inflamed lungs. Also, TNF-alpha-induced lung inflammation is not sufficient to drive murine arthritis

Mislocalization of the interferon inducible protein IFI16 by environmental insults: implications in autoimmunity

The nuclear DNA sensor IFI16, a member of PYHIN family of proteins, was previously studied for its role in cell cycle regulation, tumor suppression, apoptosis and DNA damage signaling. Autoantibodies against IFI16 are prevalent in the sera of patients with systemic autoimmunity, thus depicting physiological significance as an autoantigen. At present, the nuclear IFI16 protein has been thoroughly investigated for its role as an innate immune sensor involved in inflammasome signaling and viral restriction. While the sub-cellular localization of IFI16 during such events has been known, very little knowledge about its presence and significance in the extracellular space is available. Recently our group has discovered the presence of circulating IFI16 in the sera from systemic autoimmune patients indicating that in this setting it may be mislocalized form its nuclear site and secreted in the extracellular milieu. In this review, we will discuss the leakage of endogenous IFI16 that has been experimentally proved using in vivo and in vitro models. Also we will comment on the significance of mislocalized inflammasome components in the extracellular space and how it can be responsible for chronic inflammation

Relative efficiencies of peptidylarginine deiminase 2 and 4 in generating target sites for anti-citrullinated protein antibodies in fibrinogen, alpha-enolase and histone H3.

OBJECTIVE:Peptidylarginine deiminase 2 (PAD2) and PAD4 are expressed in the synovium of rheumatoid arthritis (RA) patients and catalyze citrullination of arginine residues in proteins targeted by anti-citrullinated protein antibodies (ACPAs). Little is known about the relative importance of PAD2 and PAD4 in generating citrullinated self-antigens. Here we investigate the ability of PAD2 and PAD4 to generate citrullinated targets for ACPAs in four human proteins. METHODS:Synovial fluid (SF) and plasma were collected from 42 RA patients. Human fibrinogen, human alpha-enolase (ENO1), human histone H3, and human serum albumin (HSA) were citrullinated in vitro by PAD2 or PAD4. The total degree of citrullination was determined using the anti-modified citrulline approach. Antibody binding to native and citrullinated proteins was measured by ELISA. RESULTS:ACPAs within pooled SF from multiple RA patients reacted equally well with, and cross-reacted with, PAD2- and PAD4-citrullinated fibrinogen. ACPAs from most individual patient SF and plasma samples bound equally well to PAD2- and PAD4-citrullinated fibrinogen or ENO1. When histone H3 was used as target, PAD4 was generally superior in generating epitopes recognized by ACPAs. No binding to citrullinated HSA was observed. CONCLUSION:In most patients, PAD2 and PAD4 are equally efficient in generating citrullinated target sites for ACPAs in fibrinogen and ENO1. The binding of autoantibodies to histone H3 was generally higher after citrullination with PAD4 than with PAD2. Citrullinated HSA is not a target for ACPAs