Quantitative Disruption of the NET–ACPA–IL-6 Autoimmune Amplification Loop by Anti-NETotic Agents in Collagen-induced Arthritis

Abstract

BACKGROUND: Rheumathoid arthritis (RA) is a condition characterized by inflammation of joint lining, which leads to joint destruction and loss of function. Neutrophil extracellular traps (NETs), anti-citrullinated protein antibodies (ACPAs), and interleukin (IL)-6 have been determined to play key roles in development and progression of RA. NETs may trigger citrullination and autoantigen exposure that can lead to ACPA production, while IL-6 increases tissue damage and inflammatory responses. This linked pathway suggests an autoimmune amplification network, but their relationship with disease progression and severity remains unclear. Therefore, this study was conducted to evaluate the relationship of this pathway in collagen-induced arthritis (CIA) mice.

METHODS: Male albino mice were randomly divided into six groups, comprised of healthy control, acute CIA, chronic untreated CIA, and CIA treated with either Myricetin, RL6, or CI-amidine. Levels of serum myeloperoxidase (MPO)-DNA, neutrophil elastase (NE)-DNA, citrullinated histone H3–neutrophil elastase complexes (CitH3–NE), IL-6, IL-1β, tumor necrosis factor-alpha (TNF-α), and ACPAs were determined using enzyme-linked immunosorbent assay (ELISA). These parameters were then statistically analyzed.

RESULTS: Untreated chronic CIA resulted in the highest level of ACPA (81.87±6.17 ng/mL) and had the strongest positive correlation between IL-6 and ACPAs (r=0.74, p<0.001). All treatments normalized CitH3–NE levels. Myricetin was the most effective in reducing TNF-α by 88.4% and abolishing the significant IL-6–ACPA correlation. This indicated a substantial reduction in the association between inflammatory and autoimmune biomarkers.

CONCLUSION: The results demonstrate a NET–ACPA–IL-6 amplification network in CIA and provide quantitative evidence for anti-NETotic therapies that can differentially dismantle this network. Correlation-network profiling may be a novel pharmacodynamic approach for the assessment of therapeutic efficacy in RA.

KEYWORDS: neutrophil extracellular traps, NETosis, anti-citrullinated protein antibodies, IL-6, collagen-induced arthritis, PAD4 inhibition, MPO inhibition