Single-Nucleus and Bulk RNA Sequencing Reveals the Involvement of Natural Killer and CD8+ T Cells in the Progression of Androgenetic Alopecia.

Abstract

Background: Androgenetic alopecia (AGA) is the most common type of androgen-associated hair loss. Emerging evidence highlights inflammation as a critical mediator in follicular miniaturization and disease progression. This investigation systematically explores inflammatory mechanisms in AGA through comprehensive analysis of hair follicles transcriptional profiles combined with cellular heterogeneity.

Methods: Matched follicular specimens were procured from AGA patients: occipital non-balding units (controls) versus frontal alopecic zones (experimental). Bulk RNA-sequencing was conducted on Norwood-Hamilton grade 3-5 AGA scalp tissues to delineate inflammatory signatures. Subsequent single-nucleus RNA sequencing (snRNA-seq) of grade 5 specimens resolved cellular heterogeneity. Immune subsets (NK/CD8⁺ T cells), vascular endothelia (BECs), keratinocytes, and fibroblasts were transcriptionally characterized. Findings were validated through immunofluorescence cytochemistry (IFC) and reverse transcription quantitative PCR (RT-qPCR).

Results: Bulk RNA-sequencing of AGA hair follicles revealed heightened inflammatory signatures in grade 5 patients compared to grade 3-4 counterparts. To dissect cellular heterogeneity, we systematically investigated the dynamic changes of immune cells in hair follicles of AGA patients using snRNA-seq technology for the first time. The result showed that grade 5 AGA hair follicles, identifying significant enrichment of natural killer (NK) and CD8⁺ T cells in balding hair follicles. Concurrently, blood endothelial cells (BECs) in balding follicles exhibited downregulation of angiogenesis-related genes. Notably, IL-15-a cytokine critical for NK/CD8⁺ T cell proliferation-was overexpressed in BECs, keratinocytes, and fibroblasts, suggesting a microenvironmental cue for immune cell expansion.

Conclusion: These findings collectively implicate NK and CD8⁺ T cell infiltration as drivers of inflammatory exacerbation in AGA. By blocking IL-15 signaling-mediated immune activation may be an innovative therapeutic approach to promote hair regeneration in AGA patients.