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

Haijing Fu,1 Wumei Zhao,1 Leiwei Jiang,2,3 Shijun Shan1,4 1Department of Dermatology, Xiang’an Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, 361000, People’s Republic of China; 2Department of Dermatology, Guizhou Provincial People’s Hospital, Guiyang City, Guizhou Province, People’s Republic of China; 3GuiYang First People Hospital, Guiyang, 550002, People’s Republic of China; 4College of Mathematical Medicine, Zhejiang Normal University, Jinhua, 321004, People’s Republic of ChinaCorrespondence: Shijun Shan, Email [email protected] Leiwei Jiang, Email [email protected]: 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.Keywords: androgenetic alopecia, natural killer cells, CD8+ T cells, inflammation and snRNA-seq