160. Testosterone Shifts CD301b+ Tissue-Resident Macrophage Homeostasis Towards an Anti-Viral Response That Inhibits Cutaneous Repair

PURPOSE: Our previous human and animal studies have consistently established a strong link between testosterone (T) and dysregulated wound healing (WH) and scarring. Older mouse research suggested that T delayed wound healing by elevating TNFa, promoting pro-inflammatory monocyte and macrophage recruitment from peripheral blood, which would contradict T’s typical anti-inflammatory roles. Conversely, our prior work revealed that T’s impact on wound healing (WH) is associated with early anti-inflammatory effects linked to CD301b+ macrophages. In this study, we hypothesized that the effects of T might differ in peripheral blood-derived CD301b+ macrophages that appear in the mid-phases of WH versus tissue-resident CD301b+ macrophages in healthy skin. METHODS: Male and female C57BL/6 mice underwent gonadectomy, with half receiving subcutaneous testosterone (+T) twice weekly. All animals underwent four 6 mm wounds for at least 14 days post-castration. Unwounded skin samples were collected on POD 0. Mannosylated-clodrosome were used to deplete CD206+ [mannose receptor] macrophages (typically co-expressed with CD301b). Mice underwent local intradermal administration of m-Clodrosome at PODs -2 and 0 versus systemic retro-orbital injections at PODs 0 and 2 versus control. At PODs 1 and 5, wound tissue samples were processed for flow cytometry, Luminex assay, histology, and qPCR analysis. RESULTS: As anticipated, POD1 +T wounds showed a robust presence of CD301b+CD206+ macrophages in comparison to noT. Successful depletion was confirmed with flow cytometry analysis of m-Clodrosome-treated mice showing a marked reduction of CD206+ populations. Interestingly, +T unwounded skin also displayed a significantly higher CD301b+ CD206+ population when compared to intact noT skin. Further UMAP clustering revealed four distinct CD301b+ macrophage and dendritic cell (DC) signatures in +T normal skin. Clusters 4 and 5, enriched in +T, co-express CD64+, F4/80+, CD206+, CD163+, IL-27+ and IFNg+ markers. Anti-viral signatures were increased on flow cytometry (e.g., IFNg) and scRNA-seq (e.g., Mx1). Tissue-resident CD206+ macrophage depletion with local injection of m-Clodrosome pre-wounding abolished T-induced WH impairment. In contrast, systemic administration at the time of wounding to eliminate peripheral blood-recruited CD206+ macrophages further worsened +T wound repair. CONCLUSION: Surprisingly, testosterone treatment appears to alter the baseline tissue-resident macrophage phenotype in healthy skin, as evidenced by increased CD301b+ DC and macrophage populations in unwounded mouse skin. In our study, localized depletion of macrophages using liposomal clodronate targeting CD206-expressing cells rescued the delayed wound healing caused by testosterone, whereas systemic depletion during the inflammatory phase did not. These findings may eventually inform optimal hormone timing (i.e., immediate pre-surgical exposure being worse than post-surgical) and cell-specific therapeutic approaches to prevent T-mediated WH impairment and hypertrophic scarring. Further, they suggest that dermal resident macrophages may play a more important initial role in directing the subsequent fate of wound repair than previously appreciated.