Neutrophil extracellular traps exert both pro- and anti-inflammatory actions in rheumatoid arthritis that are modulated by C1q and LL-37

Publication date: Available online 28 January 2019

Source: Journal of Autoimmunity

Author(s): Matthieu Ribon, Sarra Seninet, Julie Mussard, Mireille Sebbag, Cyril Clavel, Guy Serre, Marie-Christophe Boissier, Luca Semerano, Patrice Decker

Abstract

Objective

Neutrophil 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.

Methods

NET 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.

Results

PMN 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.

Conclusion

We 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.

Graphical abstract

Dual activity of NET. Activated PMN enter NETosis in response to some stimuli. In RA, PMN are significantly more sensitive to NETosis triggering. These NET directly activate (pink arrows) steady state PMN (left) and macrophages (MΦ, right), leading to strong IL-8, TNF and IL-6 secretion (three important cytokines in RA) but low IL-10 induction, i.e. a pro-inflammatory profile. In addition, RA NET are significantly more stimulatory than normal NET. Although NET are recognized by ACPA, the resulting immune complexes do not clearly enhance PMN and MΦ response to NET. Likewise, endosomal TLR are probably not involved in NET sensing. Regarding MΦ, NET-induced activation is specifically increased in the presence of DNA-binding molecules like C1q and LL-37 (blue arrows). C1q in combination with LL-37 carry NET to PMN and MΦ which express surface C1q and LL-37 receptors (C1qR, LL-37-R). Because MΦ express much more C1qR than PMN, MΦ (and not PMN) activation by NET is enhanced by C1q or C1q/LL-37. On the contrary, NET induce an anti-inflammatory profile in strongly-activated MΦ but not PMN (green arrows). Indeed, NET specifically (partly) inhibit secretion of the pro-inflammatory cytokine IL-6 by MΦ in response to LPS, especially in the presence of both C1q and LL-37, and inversely enhance IL-10 secretion by those MΦ.

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