Researchers have developed an approach for discriminating between neutrophil extracellular traps (NETs) released by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)-dependent and NOX-independent mechanisms. “We discovered that distinct disease groups have mainly either NOX-dependent or NOX-independent NETs,” explains Johan van der Vlag, corresponding author of the new study.

As a potential source of autoantigens, NETs are implicated in the pathogenesis of a number of autoimmune diseases. These structures, which are composed of DNA with histones and granule enzymes (such as myeloperoxidase (MPO) and neutrophil elastase), are released by neutrophils in response to various stimuli.

Credit: Springer Nature Limited

NOX-dependent NET formation involves the assembly of NOX and subsequent generation of reactive oxygen species, culminating in lytic release of NETs, whereas NOX-independent NETs are released through blebbing of the nuclear envelope. Current methods for quantifying NET formation in vivo measure MPO and DNA, but do not distinguish between NETs produced by NOX-dependent and NOX-independent pathways.

“We discovered that the N-terminal histone tails were cleaved off in NOX-dependent NETs by neutrophil elastase, but not in NOX-independent NETs,” remarks van der Vlag. Given these findings, the researchers developed sandwich ELISAs that employed antibodies against either the N-terminal tails or C-terminal regions of histones as the capturing antibodies, and anti-MPO antibodies as the detecting antibodies. ELISAs employing antibodies against the N-terminal histone tails could detect NOX-independent NETs but not NOX-dependent NETs, whereas ELISAs that employed antibodies against the histone C-terminal region could detect both NOX-dependent and NOX-independent NETs equally.

To ascertain the in vivo relevance of NOX-dependent and NOX-independent NET production, they measured levels of NETs in the plasma of patients with various NET-associated diseases. In patients who were positive for MPO–DNA complexes, most of those with psoriatic arthritis tested negative for NOX-independent NETs, whereas the majority of patients with either rheumatoid arthritis or systemic lupus erythematosus tested positive.

In vitro, NOX-independent NETs had greater immunostimulatory effects on endothelial cells than NOX-dependent NETs and induced the expression of various adhesion markers and pro-inflammatory cytokines.

Going forward, van der Vlag’s group plan to apply this approach to measure NOX-dependent and NOX-independent NETs in large cohorts of patients with rheumatic diseases, develop strategies that specifically interfere with NOX-dependent or NOX-independent NET formation, and elucidate the mechanisms underlying NET-mediated activation of endothelial cells.