ABSTRACT: Background: Alopecia areata (AA) is an autoimmune hair loss disorder characterized by follicular destruction. While bulk transcriptomic studies have identified contributory inflammatory axes, specific cell pathways remain underexplored. Objective: To characterize the single-cell transcriptomic landscape underpinning AA scalp compared to healthy controls. Methods: We performed single-cell RNA-sequencing on lesional and nonlesional scalp biopsies from 13 patients with moderate-to-severe AA (5 with alopecia totalis/universalis [AT/AU]) and 11 healthy controls. Results: Overall, we profiled 41,067 high-quality cells. Lesional AA samples demonstrated robust Th1 activation and cytotoxicity, with upregulated IFNG, GZMH/K, and XCL1/2. Concurrently, Th2 skewing (IL13, IL13RA1, IL4R) and TNFRSF4/OX40 elevations in lesional CD4+ and regulatory T-cells were also observed. IL15, JAK2/3, and STAT1 levels were increased in distinct lesional dendritic cell subsets, with JAK/STAT genes also upregulated in fibroblasts and keratinocytes. Fibroblasts and smooth muscle cells exhibited enriched pro-inflammatory and pro-fibrotic markers (CXCL9, CCL26, POSTN, COL5A3, COL6A6). Lesional keratinocytes further demonstrated downregulated hair keratins and increased interferon signaling. AA endothelial cells showed increased angiogenic and interferon signatures. Compared to patchy AA and controls, AT/AU demonstrated higher expression of multiple cytotoxic, Th1, Th2, and JAK/STAT markers in immune cells, and proliferative and inflammatory signatures in non-immune cells. Conclusion: This comprehensive high-resolution single-cell map uncovers potential communication networks between immune and non-immune cell populations in AA scalp, possibly disrupting immune privilege at the hair follicle and driving disease progression and/or severity. Clinical Implications: These data provide single-cell-resolution insights into AA pathogenesis and potential molecular targets for precision therapies tailored to AA severity.
