Bulk Transcriptome and Single‐Cell Sequencing Datasets Reveal Key Programmed Cell Death Patterns in Radiation‐Induced Lung Injury: Crosstalk Between NETosis and Macrophages' Responses

Programmed cell death (PCD) has been implicated in the development and progression of radiation-induced lung injury (RILI). However, the specific PCD subtype most relevant to RILI remains unclear. We integrated bulk transcriptome datasets (GSE25295, GSE41789) and a single-cell RNA sequencing dataset (GSE211713) from lung tissue to analyze the cell type-specific PCD patterns in RILI. Differentially expressed genes (DEGs) were identified using limma for bulk data and Seurat for scRNA-seq data. PCD-related DEGs were extracted based on curated gene sets, followed by functional enrichment analyzes. Intercellular communication was assessed using CellChat, and transcription factor (TF) regulatory networks were constructed using RcisTarget and AUCell. Bulk transcriptome analyzes revealed 465 radiation-responsive genes enriched in inflammatory and immune pathways. Among multiple PCD subtypes, NETosis, a specialized form of neutrophil-driven PCD characterized by the release of neutrophil extracellular traps (NETs) composed of DNA and granule proteins, was the most significantly enriched in irradiated lung tissue (FDR < 0.05). Neutrophils showed the highest NETosis scores, accompanied by activation of MAPK, IL-17, TNF, and TLR signaling. CellChat analysis revealed enhanced bidirectional communication between neutrophils and AM1/IM1 through SPP1-CD44, ANXA1-FPR, and CCL/CXCL signaling. NETosis strongly correlated with M2 polarization, and key TFs (Mafg, Mxd4, Klf4, Max, Cux1, and Irf9) were identified as putative upstream regulators driving M2 macrophage polarization and neutrophil activation. Our findings highlight NETosis, mediated by enhanced neutrophil-macrophage crosstalk and TF-driven immune polarization.