Day-7 single-cell RNA sequencing of acute lung responses after 17-Gy whole-thorax irradiation in mice.

e24193 Background: Radiation pneumonitis (RP) is a clinically important toxicity of thoracic radiotherapy and can set the stage for later fibrotic remodeling. The early post-irradiation window is attractive for intervention, but the multicellular biology of acute RP is not well resolved. We used day-7 single-cell RNA sequencing (scRNA-seq) in a murine RP model to define actionable pathways and potential biomarkers relevant to radiation oncology. Methods: Ten-week-old male C57BL/6 mice received single-fraction 17 Gy whole-thorax irradiation (IR) or sham control (NC). Day-7 micro-CT confirmed pneumonitis-like opacities. Lungs were processed for 10x Genomics scRNA-seq (OeBio; IR n = 3, NC n = 3). After filtering doublets/low-quality cells, 7,587–9,040 cells per sample were retained (mean UMI/cell 6,082–6,916; mean genes/cell 2,099–2,107; mitochondrial UMI fraction 0.0262–0.0329). Integrated analysis yielded 17 clusters spanning T cells, neutrophils, monocytes, macrophage lineages (including alveolar macrophages, AMs), Dendritic cells (DCs), B cells, Natural killer (NK) cells and stromal/epithelial compartments. Differential expression with GO/KEGG enrichment was performed. Results: By day 7, IR shifted the lung toward innate inflammation: neutrophils and monocytes increased, while T/NK and B cells decreased; AMs were markedly reduced. Across myeloid subsets, enrichment converged on TNF/NF-κB, NOD-like receptor, IL-17 signaling, and oxidative-stress programs. Neutrophils showed a lipid-mediator shift (Alox5ap up; Ptgs2 and Acod1 down), and monocytes upregulated acute-phase and lipid-inflammatory genes (Saa3, Pla2g7). Tissue compartments demonstrated parallel injury responses: Type II alveolar epithelial (AT2) cells increased DNA damage/senescence markers (Cdkn1a) with inflammatory response modules, and endothelium showed leukocyte-recruitment and inflammatory/Type I IFN features (Icam1 up). Antigen processing/presentation programs were reduced in AMs and AT2 cells (Cd74/MHC-II down). In lymphoid subsets, γδT17/ILC signatures were enriched for stress-associated pathways, supporting an IL-17–linked inflammatory niche with weakened adaptive immune crosstalk. Conclusions: Acute RP at day 7 after 17 Gy whole-thorax irradiation is characterized by a coordinated IL-17/NOD–Type I IFN injury program, myeloid lipid-mediator rewiring, and early loss of antigen-presentation capacity in AMs and AT2 cells. These data highlight tractable mitigation directions (IL-23/IL-17 signaling, leukotriene-related pathways, and myeloid recruitment) and nominate translational candidates (SAA, PLA2G7, IL-17–associated mediators) for early risk stratification.