Multi-Omics Profiling Reveals Neutrophil Extracellular Trap Dysregulation in Diabetic Foot Ulcer Healing Impairment

Diabetic foot ulcer (DFU) affects approximately 25% of diabetic patients and represents the leading cause of non-traumatic lower extremity amputation. Neutrophil extracellular traps (NETs) contribute to chronic inflammation; however, their mechanistic role in DFU healing failure remains incompletely characterized. This study integrated bulk RNA sequencing (GSE143735, n = 9) and single-cell RNA sequencing (scRNA-seq; GSE165816, n = 11) datasets to investigate NET-related transcriptional programs. Differential expression analysis identified 96 differentially expressed genes, with significant NET pathway enrichment in non-healers (normalized enrichment score = 4.35, false discovery rate q < 0.001). Analysis of 33,654 single cells revealed elevated NET activity scores in neutrophils from non-healing wounds (p = 4.73 × 10−159). Four neutrophil subpopulations were identified, with the NETs-high subset expanded in non-healers (43.1% versus 15.4%). Cell–cell communication analysis demonstrated enhanced S100A8/A9–RAGE and IL1B–IL1R signaling in the non-healing state. A six-gene signature (S100A8, S100A9, MPO, ELANE, NCF1, HMGB1) achieved an area under the receiver operating characteristic curve of 0.750 for healing prediction under leave-one-out cross-validation. These findings implicate NET pathway activation as a potential driver of DFU healing impairment and identify candidate prognostic biomarkers warranting prospective validation.