Background/Purpose: Activated neutrophils (PMN) form neutrophil extracellular traps (NET), fibers of DNA and associated proteins expelled in the extracellular space. Increased NET formation has been reported in rheumatoid arthritis (RA). Moreover, macrophages play a key role in RA pathogenesis. We have previously shown that NET are pro-inflammatory on resting non-polarized macrophages, whereas NET partly inhibit the response of LPS-stimulated macrophages (Ribon et al., J. Autoimmun, 2019, 98:122). We now aimed to characterize inflammatory properties of NET on polarized macrophage sub-populations in RA in comparison to healthy donors (HD) and to determine the mechanisms and pathways involved.

Methods: Primary blood PMN/monocytes were freshly purified from HD and RA patients. Monocytes were differentiated into non-polarized (M0), anti-inflammatory (M2a, M2c) or pro-inflammatory (M1) macrophages with cytokines. NET were induced in vitro by stimulating PMN with PMA and characterized. Mouse (wild-type, TLR9-deficient or C1q-deficient) bone marrow cells were used to differentiate macrophages and to prepare NET from purified PMN. Macrophages were cultured with NET in the presence/absence of LPS or an aryl hydrocarbon receptor (AhR) antagonist. Cell purity, phenotype and activation were estimated by flow cytometry. Cytokine secretion was measured by ELISA. The pathway triggered was estimated by bulk RNA-seq. Gene expression was confirmed by qRT-PCR.

Results: All resting macrophage subpopulations were activated by NET, leading to a pro-inflammatory response. The pro-inflammatory cytokine IL-8 was secreted, whereas secretion of the immunomodulatory IL-10 was minimal. Even M2 macrophages were activated toward this pro-inflammatory profile, with a stronger response in RA patients. In response to LPS, NET inhibit IL-6 secretion in HD macrophages whereas RA M1 and M2a macrophages were resistant to this anti-inflammatory activity of NET. Moreover, RA M1 macrophages produce more IL-8. In mouse macrophages, TLR9 is not involved in NET recognition. Moreover, C1q loaded on NET is not necessary to trigger macrophage activation. RNA-sequencing confirmed the induction of pro-inflammatory mediators, whereas IL-10 was down-regulated, and suggested that the AhR pathway is involved in macrophages in response to NET via CYP1A1 induction. Triggering of the AhR pathway by NET was next confirmed by culturing M1 macrophages with RA NET in the presence of an AhR antagonist, which showed inhibition of CYP1A1 expression.

Conclusion: Pro-inflammatory activities of NET clearly dominate anti-inflammatory activities in macrophages, particularly in RA patients, suggesting a pathogenic role of NET in RA by inducing a pro-inflammatory response of macrophages through AhR.

« Back to ACR Convergence 2022

ACR Meeting Abstracts - https://acrabstracts.org/abstract/neutrophil-extracellular-traps-net-trigger-an-enhanced-pro-inflammatory-response-in-macrophage-subpopulations-in-rheumatoid-arthritis-patients-via-the-aryl-hydrocarbon-receptor-ahr-pathway/