Integrated Analysis of Macrophage-Associated Necroptosis in Alopecia Areata.

Necroptosis, a form of programmed cell death, promotes inflammation in immune-mediated diseases, but its role in alopecia areata (AA) remains unclear. In this study, we investigated necroptosis-related signatures in AA using integrated transcriptomic and histological analyses. Four bulk RNA-seq datasets from the GEO database, comprising 191 scalp samples from AA patients and healthy controls, were analysed by ssGSEA and GSEA to assess necroptosis-related signatures. To address the cellular heterogeneity inherent in bulk RNA data, we further performed single-cell RNA-sequencing (scRNA-seq) to investigate cell-specific mechanisms, and immunofluorescence staining of scalp biopsies was conducted to validate the expression and cellular localisation of key necroptosis-related markers. Necroptosis-related scores were significantly higher in AA than in controls, correlated with disease severity and decreased after JAK/TYK2 inhibitor therapy. At single-cell resolution, a macrophage subset (IL4I1+ macrophages_1) showed the strongest necroptosis-related signal and was associated with enhanced macrophage-fibroblast communication involving TGFβ, ITGB2 and GAS6 signalling, suggesting perifollicular microenvironmental remodelling. Immunofluorescence further supported increased necroptosis-associated signalling and macrophage enrichment in AA lesions. Together, these findings suggest that macrophage-associated necroptosis-related programmes may represent a disease-associated inflammatory component in AA and support further mechanistic investigation of necroptosis-associated pathways in this disease.