Neutrophil extracellular traps mediate macrophage polarization and pyroptosis through the TLR9-NF-κB/AIM2 pathway exacerbating myositis-associated interstitial lung disease.

BACKGROUND

Previous studies by our research group have demonstrated that neutrophil extracellular traps (NETs) play a pathogenic role in myositis-associated interstitial lung disease (IIM-ILD). Based on this, we hypothesized that NETs may contribute to the pathogenesis of IIM-ILD by regulating macrophage polarization and pyroptosis.

METHODS

Pathological studies were conducted using lung biopsy samples from a dermatomyositis-associated interstitial lung disease (DM-ILD) patient and lung tissues from experimental autoimmune myositis (EAM) mice. The NETs formation inhibitor Cl-amidine was administered to intervene in EAM mice, followed by pathological validation. In vitro, phorbol ester-induced THP-1 cells were stimulated with NETs. Morphological changes in cells were observed via transmission electron microscopy (TEM). Western blot (WB), immunofluorescence, and flow cytometry were used to detect macrophage polarization and pyroptosis markers. Enzyme-linked immunosorbent assay (ELISA) was employed to measure inflammatory cytokine levels.

RESULTS

In lung tissues from a DM-ILD patient and EAM mice, CD11b and CD80 expression significantly increased, indicating extensive infiltration of monocyte-derived macrophages and polarization toward the M1 phenotype. Cl-amidine intervention in EAM mice markedly reduced NETs infiltration, suppressed TLR9 and AIM2 activation, and inhibited M1 polarization and pyroptosis in THP-1 cells. Transcriptome sequencing and WB results revealed elevated expression of TLR9, AIM2, IL-1β, and TGF-β. Compared to controls, NETs-stimulated THP-1 cells exhibited M1 polarization and upregulated expression of AIM2 inflammasome and pyroptosis markers, including GSDMD and N-GSDMD.

CONCLUSION

This study elucidates the critical role of the NETs-TLR9/AIM2-macrophage axis in IIM-ILD, offering experimental evidence to support therapeutic strategies targeting NETs.