Human hair–bearing skin organoids reveal cell type–specific susceptibility and innate immune responses to herpes simplex virus 1

Epithelial surfaces are the primary sites of lytic herpes simplex virus 1 (HSV-1) infection following initial exposure or reactivation from latency. To model this complex environment, we used human hair–bearing skin organoids and combined single-cell spatial transcriptomics with imaging to map viral spread and host responses across skin architecture. Our data suggest delayed progression of the HSV-1 life cycle in epidermal keratinocytes and hair follicle cells compared to the dermis. Infection triggered spatially organized host responses, including induction of IER3 in papillary fibroblasts. In basal keratinocytes, IRF3 was up-regulated in both infected and neighboring uninfected cells, consistent with paracrine activation of antiviral signaling. Inflammatory responses were compartmentalized in a cell type–specific manner, with TNFSF9 (4-1BBL) induced in papillary fibroblasts and tumor necrosis factor–α (TNF-α) induced in keratinocytes. Enzyme-linked immunosorbent assays confirmed secretion of these cytokines and the TNF-α–responsive chemokines CXCL2 and CXCL10. CXCL2 expression was enriched in cells adjacent to TNF-α–expressing cells, highlighting spatial coordination of inflammatory signaling. While interferon-β reduced viral replication, TNF-α and 4-1BBL did not, suggesting that their primary role is to orchestrate immune cell recruitment and activation. These findings establish human hair–bearing skin organoids as a physiologically relevant model for investigating spatial host–HSV-1 interactions.