Integration analysis of transcriptomics revealed NETosis heterogeneity and Ncf1 as a prognostic biomarker in...

Neutrophil extracellular traps (NETs) contribute to neutrophilic asthma (NA) pathogenesis, but systematic analyses of NETosis remain limited. This study aimed to identify NETs-associated biomarkers and explore their clinical relevance in NA.

A murine NA model was established through OVA/CFA sensitization. Transcriptomic profiling of lung tissues identified NETs-related differentially expressed genes (DEGs), these were then refined via LASSO regression and validated through in vivo functional assays.

NA mice exhibited pronounced inflammatory cell infiltration and collagen deposition surrounding the trachea, bronchus and blood vessel. Also, they had increased total numbers of immune cells, neutrophils, and higher expression of IgE, IL-8, TNF-α and MPO in serum and BALF. They also did not respond well to dexamethasone treatment. RNA-seq revealed 667 DEGs, with pathway enrichment in NETs formation. Elevated levels of NETs components (Cit-H3/NE/MPO/PADI4), cfDNA in BALF and serum, and ROS in BALF were also confirmed in NA mice. Then, 19 genes associated with NETs were selected and validated by RT-qPCR. LASSO regression and ROC analysis prioritized NCF1 as a pivotal NETs biomarker, showing strong diagnostic accuracy. Together with NCF1/MPO co-localization study by immunofluorescence, NCF1 immunohistochemistry confirmed their utility as diagnostic biomarkers for NETs-related pathologies. We predicted that macrophages were highly expressed in NA and positively correlated with neutrophils, and double immunofluorescence labeling analysis of LY6G and F4/80 suggested the correlation of them.

Our multi-modal analysis identifies NCF1-driven NETosis and myeloid cell interactions as key factors influencing NA development and glucocorticoid resistance, providing actionable targets for therapeutic intervention.