CSF2 drives radiation-induced lung injury by eliciting PADI4-high pathogenic neutrophils

Radiation-induced lung injury (RILI) represents a severe, therapy-refractory complication of thoracic radiotherapy. Although neutrophils have been implicated in RILI pathogenesis, the specific pathogenic subsets and the molecular mechanisms governing their recruitment and functional specialization remain elusive. This study aims to decode the crosstalk between damaged epithelial cells and neutrophils in the RILI microenvironment.

We established a murine RILI model using 17 Gy thoracic irradiation and performed integrated single-cell RNA sequencing (scRNA-seq) and bulk transcriptome profiling to map the immune landscape. Neutrophil heterogeneity and developmental trajectories were analyzed using pseudotime algorithms. Mechanistic investigations used an indirect co-culture system of irradiated lung epithelial cells and HL-60 neutrophil-like cells, recombinant CSF2, CSF2-neutralizing antibodies, and in vivo anti-GM-CSF blockade. The therapeutic potential of PADI4 inhibition was evaluated using GSK484 in RILI mice, followed by histopathological, molecular, immunofluorescence, ELISA, and bulk RNA-seq analyses.

Integrated multi-omics analyses identified PADI4 as a central neutrophil-associated molecule in RILI, with preferential enrichment in immature, NETosis-prone neutrophil subsets mobilized during emergency granulopoiesis. Among PAD family members, PADI4 showed the most prominent neutrophil-restricted and injury-responsive expression pattern. Mechanistically, radiation-damaged epithelial cells secrete CSF2, which induces a PADI4-high activated myeloid phenotype in vitro; conversely, CSF2 neutralization attenuated this response. In vivo GM-CSF blockade reduced PADI4-positive neutrophil infiltration and lung injury severity. Pharmacological PADI4 inhibition with GSK484 suppressed citrullinated histone H3 generation, NETosis-associated programs, neutrophil chemotaxis modules, and tissue damage in the RILI model.

This study delineates an epithelial-myeloid damage-response circuit in RILI and identifies CSF2-driven PADI4 activation in pathogenic neutrophils as a therapeutically actionable mechanism.