Squaric Acid Dibutylester Promotes Innate Immune-Driven Hair Growth with CD206⁺ Macrophage Accumulation in the Dermis

Alopecia areata (AA) is an autoimmune skin disorder that causes hair loss. Squaric acid dibutylester (SADBE) is used for AA treatment as a topical immunotherapy that promotes hair growth by inducing allergic contact dermatitis in skin lesions. However, the mechanism of action remains unclear. C3H/HeJ mice spontaneously develop AA, and SADBE application induces hair growth at the lesion on day 28. In healthy young mice treated with SADBE, hair growth was observed after day 14. Fluorescent immunostaining of SADBE-treated skin tissues revealed a remarkable accumulation of macrophages in the dermis on day 3. These macrophages were divided into 3 subsets that formed a layered structure; in particular, CD206⁺F4/80⁺ cells were localized near the dermal papilla. Flow cytometric analysis also showed these 3 subsets in SADBE-treated skin on day 3. Macrophage depletion by intradermal clodronate injection inhibited SADBE-induced hair growth, suggesting macrophage dependency. A single SADBE application induced hair growth without sensitization, indicating that the acute inflammation mediated by innate immune cells was sufficient. Hair growth by repeated SADBE application in AA-affected mice closely correlated with the increase of CD206⁺ macrophages. These findings suggest that innate immune cells, particularly macrophages, play an important role in SADBE-induced hair growth, which would be potential targets in novel therapies for AA.