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Background: Neutrophil extracellular trap (NET) formation has been implicated as a pathogenic mechanism in both rheumatoid arthritis (RA) and interstitial lung disease (ILD). However, the role of NETs in RA-associated ILD (RA-ILD) and the mechanisms driving NET formation remain unclear. This study aimed to assess the involvement of NETs in RA-ILD and elucidate the underlying mechanisms. Methods: Single-cell sequencing was used to identify changes in the quantity and function of neutrophils in the lung tissue of a zymosan A (ZYM)-induced interstitial pneumonia arthritis model. Additionally, nuclear receptor 4A3 (NR4A3) interference was performed in HL-60 cells to assess its impact on NET formation and the transformation of MRC-5 cells into myofibroblasts. The clinical relevance of plasma myeloperoxidase-DNA (MPO-DNA), citrullinated histone 3 (Cit-H3), and cell-free DNA was evaluated in RA-ILD patients with different imaging types via a commercial enzyme-linked immunosorbent assay (ELISA). Results: In the ZYM-treated SKG mouse model, which recapitulates key features of RA-ILD, an increased population of neutrophils in the lung tissue was primarily responsible for NET formation. Mechanistically, we found that interference with NR4A3 expression enhanced NET formation in HL-60 cells, which in turn promoted the differentiation of MRC-5 cells into myofibroblasts. Clinically, plasma MPO-DNA levels are elevated in patients with RA-nonspecific interstitial pneumonia (RA-NSIP), whereas Cit-H3 levels are elevated in RA-usual interstitial pneumonia (RA-UIP) patients compared with healthy subjects. ROC curve analysis further revealed that the combination of plasma MPO-DNA, rheumatoid factor (RF), and anti-citrullinated protein (anti-CCP) and the combination of Cit-H3, RF, and anti-CCP were superior diagnostic panels for NSIP and UIP in RA-ILD patients, respectively. Moreover, compared with those from healthy controls, neutrophils from patients with RA-UIP and RA-NSIP demonstrated a significantly increased ability to form NETs and induce the differentiation of MRC-5 cells into myofibroblasts. Specifically, RA-UIP patients exhibited a greater capacity for NET formation and the differentiation of MRC-5 cells into myofibroblasts than did RA-NSIP patients. Conclusions: These findings suggest that targeting NETs may be a novel therapeutic approach for treating ILD in RA patients. [ABSTRACT FROM AUTHOR]

Plastics, combined with plasticizers, have been widely utilized worldwide. Microplastic fibers (MPFs) and dibutyl phthalate (DBP) account for the most predominant microplastics and plasticizers detected in freshwater ecosystem, with their joint toxicity being limited studied. In this study, we employed freshwater algae (Chlorella vulgaris) as toxicity test model organism to assess their growth, photosynthesis, metabolism, and oxidative response when exposing to different concentrations of polypropylene MPFs and the co-exposure of DBP. In addition, the toxic interaction between MPFs and DBP was assessed by combining the integrated toxicity value (Integrated Biomarker Response version 2, IBRv2) and the mixture toxicity index (Effect Addition Index, EAI). Our results demonstrated significant toxic effects of MPFs and DBP on C. vulgaris, and highlighted their dynamic interactions with C. vulgaris. Specifically, when combining with DBP, MPFs with high concentrations exhibited significantly increase in algae growth inhibition, photosynthetic pigment contents (Chl-a, Chl-b, and carotenoids), protein contents, and oxidative enzymes (SOD, CAT, and MDA). In terms of integrated toxicity values, higher IBRv2 values were recorded by the combined exposure of MPFs and DBP in contrast with the sole exposure groups, indicating that the combined exposure caused more severe damage to photosynthesis, oxidation and metabolism. In addition, our study recorded synergistic combined toxicity when MPFs were in high concentrations, whereas antagonistic combined toxicity when MPFs were in low concentrations. Our study highlights the MPFs concentration-dependent combined toxicity (synergistic or antagonistic) when exposing to microplastics and plasticizers in freshwater ecosystems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier B.V. All rights reserved.)