Single-cell transcriptome reveals keratinocyte subclusters that contribute to altered differentiation and inflammatory responses in atopic dermatitis

Atopic dermatitis (AD) is a common chronic inflammatory skin disease with complex, poorly understood pathogenesis due to its mechanistic heterogeneity. To investigate underlying mechanisms, we performed single-cell RNA sequencing (scRNA-seq) on lesional (LAD) and non-lesional (NAD) skin from 42 AD patients and skin from 23 healthy controls (HC). Keratinocytes (KCs) were the most abundant cell type identified. In healthy skin, KC differentiation followed a linear trajectory from basal KCs (BKs) through seven differentiated stages (DK1–DK7) to terminal keratinized cells (KK1 and KK2). In LAD, this process was disrupted, showing a reversed transition from KK2 to KK1, mainly driven by DK7. APOD and LYZ were identified as LAD-specific regulators of this aberrant keratinization, linked to IL-13- and IL-22-driven responses involving endoplasmic reticulum (ER) stress and oxidative damage. Mitochondrial and ER dysfunction were specifically enriched in LAD DK6 cells, suggesting this subcluster as a key pathogenic compartment associated with cytokine activation. Further, cell-cell interaction analysis (validated through Xenium spatial transcriptomics and immunohistochemistry) highlighted TWEAK, derived from IL13+ Th2 and cycling T cells acting on FN14+ basal and differentiated KCs, as a key contributor to AD-associated epidermal responses. These findings reveal how disrupted KC differentiation and specific immune pathways contribute to AD pathogenesis, identifying distinct cellular compartments and inflammatory circuits as potential therapeutic targets.