Objectives Neutrophil extracellular traps (NETs) act in various rheumatic diseases. Although NET formation was originally described as a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)-dependent pathway, it appears that there are also NOX-independent pathways of NET release. Currently, no tools are available that can discriminate between both NET-forming pathways. We aimed to develop a serological method allowing the discrimination between NETs generated through NOX-dependent or NOX-independent pathways.
Methods Histones from in vitro generated NOX-dependent and NOX-independent NETs were characterised with a panel of lupus-derived antibodies against N-terminal histone tails using immunofluorescence microscopy, western blot and ELISA. NETs in patients with NET-associated diseases, that is, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), psoriatic arthritis (PsA) and sepsis, were characterised in sandwich ELISAs employing antibodies against myeloperoxidase (MPO) and N-terminal histone tails as detecting and capturing antibodies, respectively. Functional responses of endothelial cells to NOX-dependent and NOX-independent NETs were assessed as well.
Results Neutrophil elastase cleaves the N-terminal tails of core histones during NOX-dependent, but not during NOX-independent NET formation. Consequently, the detection of MPO–histone complexes with antibodies against N-terminal histone tails allows discrimination between NETs formed through a NOX-dependent or NOX-independent manner. Characterisation of in vivo circulating NETs revealed the presence of NOX-independent NETs in RA, SLE and sepsis, but NOX-dependent NETs in PsA. NOX-independent NETs displayed an increased capacity to activate endothelial cells when compared with NOX-dependent NETs.
Conclusions These results indicate heterogeneity in NET-forming pathways in vivo and highlight the need for disease-specific strategies to prevent NET-mediated pathology.
- autoimmune diseases
- psoriatic arthritis
- rheumatoid arthritis
- systemic lupus erythematosus
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EP and NR contributed equally.
Handling editor Josef S Smolen
Contributors EP, NR and CY performed the experiments, interpreted the data and wrote the manuscript. JG, SB, PP, LM and MH provided patient material and wrote the manuscript. LH and JV supervised the research, interpreted the data and wrote the manuscript.
Funding This research was supported by the Radboud Institute of Molecular Life Sciences (RIMLS) PhD program and the Department of Nephrology of the Radboud University Medical Center.
Competing interests None declared.
Patient consent Not required.
Ethics approval Ethical committees of the Radboud University Medical Center and the University Hospital Erlangen.
Provenance and peer review Not commissioned; externally peer reviewed.
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