2521 - Single-Cell Profiling of Leukotriene-Related Lipid Programs in Acute Lung Injury after Whole-Thorax Irradiation

Purpose/Objective(s):

Thoracic radiotherapy (RT) can produce clinically meaningful normal lung toxicity. The first week after irradiation is a realistic window for intervention, yet the early, targetable biology of acute lung injury is still not well defined. We performed day-7 single-cell RNA sequencing (scRNA-seq) after murine whole-thorax irradiation to profile acute responses and nominate druggable lipid-mediator programs.

Materials/Methods:

Ten-week-old male C57BL/6 mice received single-fraction 17 Gy whole-thorax irradiation (IR) or sham control (NC). Day-7 micro–computed tomography (micro-CT) demonstrated pneumonitis-like opacities. Lungs underwent 10x Genomics single-cell RNA sequencing (scRNA-seq) (IR n=3; NC n=3). After removing doublets and low-quality/apoptotic cells, 7,587–9,040 cells per sample were retained (mean unique molecular identifiers [UMIs]/cell 6,082–6,916; mean genes/cell 2,099–2,107; mitochondrial UMI fraction 0.0262–0.0329). Integrated clustering yielded 17 clusters across myeloid, lymphoid, epithelial, endothelial, and stromal compartments. Differential expression and Gene Ontology/Kyoto Encyclopedia of Genes and Genomes (GO/KEGG) enrichment were conducted by major cell type.

Results:

By day 7, IR produced a clear shift toward innate inflammation: neutrophils and monocytes expanded, lymphoid populations contracted, and alveolar macrophages were markedly reduced. Neutrophils displayed a lipid-mediator shift, with Alox5ap increased and Ptgs2 and Acod1 decreased—compatible with a leukotriene-skewed inflammatory state and loss of counter-regulatory metabolic braking. Monocytes upregulated acute-phase and lipid-inflammatory genes (Saa3, Pla2g7), pointing to enhanced inflammatory lipid processing. Endothelial cells showed features of leukocyte recruitment (e.g., Icam1 up) together with enrichment of membrane lipid–related pathways. Alveolar type II (AT2) cells carried injury/stress programs (e.g., Cdkn1a up), and inflammatory signaling modules were broadly enriched across myeloid and recipient compartments.

Conclusion:

Acute lung injury at day 7 after whole-thorax irradiation is accompanied by coordinated lipid-mediator remodeling across myeloid and vascular compartments, with a leukotriene-leaning signature emerging as a plausible, druggable mitigation axis. The dataset supports follow-up testing of leukotriene-pathway inhibition with lipid-focused readouts and highlights measurable candidates (PLA2G7/SAA-linked programs and lipid mediators in blood/bronchoalveolar lavage fluid [BALF]) for early risk stratification of normal tissue toxicity.