Dysregulation of epidermal barrier in mouse skin wounds induced by sulfur mustard

Abstract

Sulfur mustard (2,2′-dichlorodiethyl sulfide, SM), is a potent chemical vesicant. Skin exposure to SM results in blistering, persistent epidermal hyperplasia, severe tissue damage, and prolonged wound repairs. SM, a bifunctional alkylating agent, causes DNA damage in mouse skin cells; however, the mechanistic action of SM in tissue injuries is still unclear. Studies showed mouse skin following SM exposure activates several stress signaling pathways including oxidative stress, and induces strong inflammatory responses. Skin structured with multilayers of keratinocytes (KCs), serves as a protection barrier against toxic chemicals. Keratins (Ks), the major structural intermediate filaments proteins in KCs, may regulate skin wound healing. In normal skin epidermis, KCs express K5 in basal cells and K10 in the differentiated suprabasal layers. Upon skin injury, KCs, activated by inflammation, express K6 and K17 to promote KCs migration for wound repair. Dysregulation of activated epidermal KCs may result in barrier breach and lead to uncontrolled inflammation and skin diseases. In addition, the cornified envelope (CE) precursor molecules, the small proline-rich (Sprr) proteins, present to protect skin from reactive oxidative species (ROS) damage. The present study was focused on SM induced alteration of skin barrier alarmin molecules in mouse ear skin. Time course studies (1-7 days) of mouse ear skin exposing to a single dose (0.08 mg) of SM showed characteristic histopathology changes over time. Significant changes of skin barrier alamin molecules (K10, K6, K17, and Sprr1B) gene expression were observed in SM injured skin post exposure. Immunohistochemistry results showed the K6 and K17 staining was restricted to the skin appendages in the naïve control, whereas samples exposed to SM had increased expression over time in the suprabasal epithelium. The activated KCs highly expressed K6, K17, and Sprr1B at the wound edge. In contrast, reduced expression of the differentiation marker, K10, indicated incompletely differentiated epithelium, suggesting weakened barrier protection in skin damaged by SM. Multiplex immunofluorescent studies showed heterogeneous coexpression of K5/ K6/K10 and K5/K10/Sprr1B in the hyperplasia behind the leading edge, suggesting hyperactivated KCs in the SM skin wounds. Overall, K10, K6, K17, and Sprr1B have value as skin wound and repair markers of SM induced injury. Understanding the molecular mechanism of action of vesicant-induced skin injury and repair involved in the dysregulation of epidermal barrier may help to identify new targets of medical countermeasures to chemical vesicant skin injury.