Circulating Neutrophil Extracellular Trap (NET)-forming neutrophils in rheumatoid arthritis exacerbation are majority dual endothelin-1/signal peptide receptor (DEspR)+ subtype.

Neutrophil extracellular traps (NETs) are associated with rheumatoid arthritis pathogenesis and severity. Since homeostatic NET-forming neutrophils [NET+Ns] have beneficial roles in defense against pathogens, their distinction from pro-injury [NET+N] subtypes is important, especially if they are to be therapeutically-targeted. Having identified circulating, pro-injury DEspR+CD11b+ [NET+Ns] in patients with neutrophilic secondary tissue injury, we determined whether DEspR+ [NET+Ns] are present in RA-flares. Whole blood samples of patients with RA-flares on maintenance therapy (n=6), were analyzed by flow cytometry (FCM) and immunofluorescence cytology followed by semi-automated quantitative confocal microscopy (qIFC). We assessed clinical parameters, levels of neutrophils and [NET+Ns], and plasma S100A8/A9. qIFC detected circulating DEspR+CD11b+ neutrophils and [NET+Ns] in RA-flare patients but not healthy controls. DEspR+ [NET+Ns] were positive for citrullinated histone H3 (citH3+), extruded DNA, decondensed but recognizable polymorphic nuclei, and [NET+N] doublet-interactions in mostly non-ruptured NET-forming neutrophils. Circulating DNA+/DEspR+/CD11b+/citH3+ microvesicles (netMVs) were observed. FCM detected increased %DEspR+CD11b+ neutrophils and DEspR+ cell-cell doublets whose levels trended with DAS28 scores, as did plasma S100A8/A9 levels. This study identifies circulating DEspR+/CD11b+ neutrophils and [NET+Ns] in RA-flare patients on maintenance therapy. Detection of circulating DEspR+citH3+ [NET+Ns] and netMVs indicate a systemic neutrophilic source of citH3-antigen concordant with multi-joint RA pathogenesis. Increased S100A8/A9 alarmin levels are associated with cell injury and released upon NET-formation. As a ligand for TLR4, S100A8/A9 forms a positive feedback loop for TLR4-induced DEspR+ neutrophils. These data identify DEspR+ neutrophils and [NET+Ns] in RA pathogenesis as a potential biomarker and/or therapeutic target.