Neutrophil Extracellular Traps–Associated RNA Impedes CD4+ Treg Differentiation by TLR7–IRF7 Axis in Ankylosing Spondylitis

Abstract

Objective

Our objective was to investigate the role of neutrophil extracellular traps (NETs) in the pathogenesis of inflammatory disorders in ankylosing spondylitis (AS).

Methods

Local and circulating NETs levels were determined by immunofluorescence (IF) and myeloperoxidase (MPO)–DNA quantification in both patients with AS and AS model SKG mice. Flow cytometry (FCM) was performed to detect the effect of NETs on CD4⁺ subpopulation differentiation. The therapeutic effects of the neutrophil elastase inhibitor sivelestat (SVT) and the peptidylarginine deiminase 4 (PAD4) inhibitor CI–amidine were evaluated in SKG mice. The localization of NETs and their ability to impede CD4⁺ Treg cell differentiation were evaluated via IF, FCM, and Western blotting. RNA sequencing and specific inhibitors were used to clarify the detailed mechanism by which NETs inhibit CD4⁺ Treg differentiation.

Results

The NETs levels were elevated locally and systemically in both patients with AS and SKG mice, which impeded the differentiation of CD4⁺ Treg cells. Blocking NETs formation via SVT or CI-amidine restored the CD4⁺ Treg ratio and subsequently alleviated inflammation in SKG mice. NETs were internalized by CD4⁺ T cells, and their associated RNA activated the Toll-like receptor 7 (TLR7)–interferon regulatory factor 7 (IRF-7) axis, which then inhibited Treg differentiation. Inhibiting CD4⁺ T cells endocytosis, removing the bound RNA component, or blocking the TLR7–IRF-7 axis abrogated the negative effect of NETs on CD4⁺ Treg differentiation.

Conclusion

Elevated NETs impeded CD4⁺ Treg differentiation by activating the TLR7–IRF-7 axis via their associated RNA in AS, and targeting NETs may be a novel treatment strategy for AS and related inflammatory disorders.