Enhanced neutrophil extracellular trap generation in rheumatoid arthritis: analysis of underlying signal transduction pathways and potential diagnostic utility

Abstract

IntroductionNeutrophil extracellular traps (NETs) have recently been implicated in a number of autoimmune conditions, including rheumatoid arthritis (RA). We examined the underlying signaling pathways triggering enhanced NETosis in RA and ascertained whether the products of NETosis had diagnostic implications or usefulness.MethodsNeutrophils were isolated from RA patients with active disease and from controls. Spontaneous NET formation from RA and control neutrophils was assessed in vitro with microscopy and enzyme-linked immunosorbent assay (ELISA) for NETosis-derived products. The analysis of the signal-transduction cascade included reactive oxygen species (ROS) production, myeloperoxidase (MPO), neutrophil elastase (NE), peptidyl arginine deiminase 4 (PAD4), and citrullinated histone 3 (citH3). NET formation was studied in response to serum and synovial fluid and immunoglobulin G (IgG) depleted and reconstituted serum. Serum was analyzed for NETosis-derived products, for which receiver operator characteristic (ROC) curves were calculated.ResultsNeutrophils from RA cases exhibited increased spontaneous NET formation in vitro, associated with elevated ROS production, enhanced NE and MPO expression, nuclear translocation of PAD4, PAD4-mediated citrullination of H3, and altered nuclear morphology. NET formation in both anti-citrullinated peptide antibody (ACPA)-positive and -negative RA was abolished by IgG depletion, but restored only with ACPA-positive IgG. NETosis-derived products in RA serum demonstrated diagnostic potential, the ROC area under the curve for cell-free nucleosomes being >97%, with a sensitivity of 91% and a specificity of 92%. No significant difference was observed between ACPA-positive and -negative cases.ConclusionsSignaling elements associated with the extrusion of NETs are significantly enhanced to promote NETosis in RA compared with healthy controls. NETosis depended on the presence of ACPA in ACPA-positive RA serum. The quantitation of NETosis-derived products, such as cell-free nucleosomes in serum, may be a useful complementary tool to discriminate between healthy controls and RA cases.

Elevated Levels of Total Cell-Free DNA in Maternal Serum Samples Arise from the Generation of Neutrophil Extracellular Traps

“…We have recently observed that cfDNA concentrations were elevated in serum, but not in plasma samples, of RA patients, when compared to similar samples from matching healthy control donors [11]. By determining that the serum cfDNA consisted of nucleosome/MPO complexes, we were able to show that the vast proportion of serum cfDNA was derived from neutrophils undergoing NETosis during the clotting procedure [11].…”

“…Consequently, NET-derived products can be detected by examining for complexes of cfDNA and neutrophil granular proteins [9]. Aberrant or excessive NET generation has been noted in a number of human pathologies, including PE [10], small vessel vasculitis [9], or rheumatoid arthritis (RA) [11]. …”

Increased levels of neutrophil extracellular trap remnants in the circulation of patients with small vessel vasculitis, but an inverse correlation to anti-neutrophil cytoplasmic antibodies during remission

“…Several of these proteins, such as histones, MPO, PR3, HMGB1 and LL37, possess proinflammatory characteristics and NETs have in vitro been shown to induce interferon (IFN)-α production in dendritic cells (5), cause epithelial and endothelial cell damage (6)(7)(8) and act as bridge between the innate and adaptive immune system (9). NETs trap and kill microbes, including bacteria, fungi and protozoa (3,(10)(11)(12)(13)(14) but have also been connected to various autoinflammatory or autoimmune diseases, such as preeclampsia, systemic lupus erythematosus (SLE), Felty´s syndrome (FS) and rheumatoid arthritis (RA) (15)(16)(17)(18)(19). NETs have also been suggested to be important in a rodent model of drug-induced vasculitis (20), implying a role of NETs in the pathogenesis of AAV as well.…”

The Contribution of Innate Immunity to the Pathogenesis of ANCA-associated Vasculitis

“…It has also been shown that the other stimulus used by us in paper I and II, TNF and ANCA, can induce NETosis (Kessenbrock et al 2009, Keshari et al 2012), but also that NETosis can occur spontaneously in unstimulated neutrophils (Sur Chowdhury et al 2014, Tang et al 2015. Although one cannot exclude that also necrotic cells are being stained with this method, it is beneficial that no NETs are being lost during the measurements.…”

“…In addition to their role as antimicrobial agents, NETs of both nuclear and mitochondrial origin have been connected to autoinflammatory or autoimmune diseases (Gupta et al 2005, Kessenbrock et al 2009, Dwivedi et al 2012, Khandpur et al 2013, Leffler et al 2013, Sur Chowdhury et al 2014, Surmiak et al 2015, Lood et al 2016, and have therefore been referred to as a double edged sword of innate immunity (Kaplan and Radic 2012).…”