• Matthieu Ribon, Sarra Seninet, Julie Mussard, Mireille Sebbag, Cyril Clavel, Guy Serre, Marie-Christophe Boissier, Luca Semerano, and Patrice Decker.
• University of Paris 13, Sorbonne Paris Cité, Bobigny, France; Inserm UMR 1125, Li2P, Bobigny, France.
• J. Autoimmun. 2019 Mar 1; 98: 122-131.

ObjectiveNeutrophil extracellular traps (NET), produced by activated polymorphonuclear neutrophils (PMN), are supposed to play a role in the pathogenesis of rheumatoid arthritis (RA), a chronic inflammatory autoimmune disease characterized by anti-citrullinated protein antibodies (ACPA). Indeed, NET contain citrullinated autoantigens and some RA autoantibodies recognize NET. However, the mechanisms by which NET trigger or perpetuate the inflammatory process in RA are hitherto not elucidated. We hypothesized that, in addition to citrullination, NET might also contain stimulatory proteins and directly activate inflammatory target cells, as PMN and macrophages.MethodsNET antigenic and inflammatory properties were analyzed in 157 healthy donors (HD) and RA patients, the largest analysis reported so far. Primary PMN and monocyte-derived macrophages were isolated and immunoglobulin G (IgG) purified. NET were induced (NETosis), isolated and quantified. NET antigenicity was analyzed by fluorescence microscopy. PMN and macrophages were stimulated with NET with/without ACPA, C1q, LL-37 or lipopolysaccharide (LPS) and cell activation was estimated by flow cytometry and ELISA.ResultsPMN from RA patients produced more NET than HD PMN. We next dissected how NET mechanistically affect inflammatory cells. Particularly, we show for the first time that RA and HD NET activated both resting macrophages and PMN, but importantly RA NET were more stimulatory, leading to secretion of inflammatory cytokines and up-regulation of HLA/CD86/CD11b. IgG from ACPA-positive RA patients specifically recognized RA and even HD NET. Nevertheless, NET-induced cell activation occurs independently of immune complex formation with ACPA. Likewise, endosomal acidification was not required. Notably, we also report that complement C1q increased the NET stimulatory activity on macrophages only, due to higher expression of C1q receptors, which was further supported by the LL-37 antimicrobial peptide. In contrast, NET specifically inhibited interleukin (IL)-6 secretion by LPS-activated macrophages and not PMN, especially with C1q/LL-37. This inhibition was not mediated by NET-derived proteases or LPS neutralization and was associated with the simultaneous induction of IL-10 secretion.ConclusionWe show that NET possess both pro- and anti-inflammatory properties depending on target cells, their activation levels and C1q/LL-37. Thus, independently of ACPA, NET modulate RA chronic inflammation via this new dual activity we identified. In addition, NET may trigger autoimmunity in RA as ACPA recognize NET antigens but not non-activated PMN. Therefore, we conclude that excess of NETosis together with enhanced NET activity participate to RA pathogenesis at different levels.Copyright © 2019 Elsevier Ltd. All rights reserved.

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