NETs accelerate aortic valve calcification by promoting M1 macrophage polarization through the TLR9 signaling pathway

Neutrophil extracellular traps (NETs) are positively correlated with the severity of calcific aortic valve disease (CAVD). This study aims to elucidate the mechanism by which NETs contribute to CAVD. The CAVD mice model was established by calcification-promoting diets, and NETs formation was modulated via intraperitoneal injection of Cl-amidine. We observed the effect of NETs on Raw264.7 cells by regulating NETs and TLR9 in vitro. Concentrations of TNF-α, MPO-DNA complex, and IL-10 were measured using ELISA. NETs formation was assessed through immunofluorescence assay citrullinated histone H3 (citH3). Expression levels of BMP2, RUNX2, IL-1β, TNF-α, IL-10, and TLR 9 were analyzed by qRT-PCR and Western blotting, while flow cytometry was used to assess the expression of CD86 and CD206 on Raw264.7 cells. Results indicated that compared to the vehicle group, the CAVD group exhibited significant valve thickening and increased calcium deposition, as well as elevated levels of inflammatory factors TNF-α and IL-1β, NET-related markers MPO-DNA complexes and citH3, ossification factors BMP2 and RUNX2, and TLR9. Conversely, IL-10 levels were significantly reduced. Cl-amidine intervention in early CAVD mice significantly improved valve thickness and reduced calcium deposition, inflammatory factors, NETs-related markers, ossification factors, and TLR9 levels, while increasing IL-10 levels. Cl-amidine may delay CAVD progression in mice by reducing NETs. In vitro studies confirmed that serum from CAVD mice induced NETs, promoting the polarization of Raw264.7 cells to the M1 phenotype via TLR9 signaling pathway, thereby releasing pro-inflammatory factors (TNF-α, IL-1β, and IL-6), and inhibiting M2 polarization and IL-10 expression. In summary, our findings suggest that NETs promote Raw264.7 cell polarization to M1 through the TLR9 signaling pathway, contributing to the inflammatory response in CAVD. This study proposes a novel therapeutic strategy targeting NETs to delay CAVD progression.