PURPOSE: Ideal wound healing (WH) requires balanced inflammation. Our prior work linked testosterone (T) treatment to undesirable WH/scarring outcomes in gender-affirming surgery. Immune cells are core modulators of wound repair and highly affected by sex hormones. We sought to identify whether changes to the immune cell repair program modulate clinical effects of T on WH.
METHODS: Castrated rats received bi-weekly testosterone (+T) or control, followed by dorsal wounding and harvest on POD5/POD10. Analysis was performed on histology, immunofluorescence, Luminex, spatial transcriptomics, and single-cell RNA-sequencing.
RESULTS: +T rats had slower wound closure at POD5 and POD10 versus controls (p<0.0001). Immunofluorescence of macrophage surface markers revealed elevated CD163+ macrophages in the +T group with changes in MIP-1α (p=0.017) cytokine levels.
6,154 cells across 8 replicates were analyzed on sc-RNA-seq. Pathway analysis showed +T macrophages displayed upregulation of TNF-α signaling via NF-Kβ and pI3K/Akt/mTOR pathways. Upregulated DEGs included pro-inflammatory and pro-angiogenic signatures, e.g., Il-1a, Tnf-a, Vegf-a. In contrast, testosterone greatly suppressed the abundance of non-CD8+ T cells on compositional analysis (p<0.001).
CONCLUSION: Testosterone uniquely alters the wound immune milieu. Increases in CD68+CD163+ macrophages and NF-Kβ activity suggest these behaviors may be mediated through macrophage shifts and suppression of adaptive T cells in favor of myeloid cells. These findings provide new insight into the complex and previously under-appreciated interplay between immune cells and sex hormones in modulating tissue repair.