Single-cell RNA sequencing reveals the impaired epidermal differentiation and pathological microenvironment in diabetic foot ulcer

Diabetic foot ulcer (DFU) is one of the most common and complex complications of diabetes, but the underlying pathophysiology remains unclear. Single-cell RNA sequencing (scRNA-seq) has been conducted to explore novel cell types or molecular profiles of DFU from various perspectives. This study aimed to comprehensively analyze the potential mechanisms underlying impaired re-epithelization of DFU in a single-cell perspective. We conducted scRNA-seq on tissues from human normal skin, acute wound, and DFU to investigate the potential mechanisms underlying impaired epidermal differentiation and the pathological microenvironment. Pseudo-time and lineage inference analyses revealed the distinct states and transition trajectories of epidermal cells under different conditions. Transcription factor analysis revealed the potential regulatory mechanism of key subtypes of keratinocytes. Cell-cell interaction analysis revealed the regulatory network between the proinflammatory microenvironment and epidermal cells. Laser-capture microscopy coupled with RNA sequencing (LCM-seq) and multiplex immunohistochemistry were used to validate the expression and location of key subtypes of keratinocytes. Our research provided a comprehensive map of the phenotypic and dynamic changes that occur during epidermal differentiation, alongside the corresponding regulatory networks in DFU. Importantly, we identified two subtypes of keratinocytes: basal cells (BC-2) and diabetes-associated keratinocytes (DAK) that might play crucial roles in the impairment of epidermal homeostasis. BC-2 and DAK showed a marked increase in DFU, with an inactive state and insufficient motivation for epidermal differentiation. BC-2 was involved in the cellular response and apoptosis processes, with high expression of TXNIP, IFITM1, and IL1R2. Additionally, the pro-differentiation transcription factors were downregulated in BC-2 in DFU, indicating that the differentiation process might be inhibited in BC-2 in DFU. DAK was associated with cellular glucose homeostasis. Furthermore, increased CCL2 + CXCL2+ fibroblasts, VWA1+ vascular endothelial cells, and GZMA+CD8+ T cells were detected in DFU. These changes in the wound microenvironment could regulate the fate of epidermal cells through the TNFSF12-TNFRSF12A, IFNG-IFNGR1/2, and IL-1B-IL1R2 pathways, which might result in persistent inflammation and impaired epidermal differentiation in DFU. Our findings offer novel insights into the pathophysiology of DFU and present potential therapeutic targets that could improve wound care and treatment outcomes for DFU patients.