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

Chanchal Sur Chowdhury,Ulrich A Walker,Sinuhe Hahn,Stavros Giaglis,Andreas Buser,Paul Hasler

doi:10.1186/ar4579

Chanchal Sur Chowdhury, Ulrich A Walker + Show 4 more

Open Access

https://doi.org/10.1186/ar4579

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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.

Highlights

Neutrophil extracellular traps (NETs) have recently been implicated in a number of autoimmune conditions, including rheumatoid arthritis (RA)
As we had previously detected significantly increased concentrations of cell-free DNA in the sera of RA patients compared with healthy controls, we were intrigued whether the provenance of this material involved NETosis [15]
RA-derived polymorphonuclear granulocyte (PMN) exhibit increased spontaneous NETosis Details of the RA study group and healthy control group are described in Table 1 and Additional file 1

Summary

Introduction

Neutrophil extracellular traps (NETs) have recently been implicated in a number of autoimmune conditions, including rheumatoid arthritis (RA). A novel feature of the biology of polymorphonuclear granulocytes (PMNs) is their ability to generate neutrophil extracellular traps (NETs) [1] via a distinct process of cell death termed NETosis [2]. A number of studies have implicated NETs in the etiology of autoinflammatory or autoimmune conditions such as preeclampsia, Felty syndrome, systemic lupus erythematosus (SLE), multiple sclerosis, and, most recently, rheumatoid arthritis (RA) [7,8,9,10,11,12,13]. Whereas the citrullinated autoantigens vimentin and α-enolase were expressed on NETs from RA PMNs, antibodies to the former were able to induce NET formation by healthy control PMNs [13,14]

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