Abstract Body (Do not enter title and authors here): Background: While macrophage to myofibroblast transition (MMT) has been reported in various tissues and diseases, it has not been studied in wound healing. We sought to identify the epigenetic mechanisms that control MMT during wound repair and if MMT is epigenetically altered in diabetic wound healing.

Methods: We used a combination of transgenic murine models, loss of function approaches in vitro and in vivo, pharmacologic inhibition in vivo, flow cytometry, spatial sequencing, and single cell RNA sequencing (scRNA-seq) in human and murine wounds.

Results: Flow cytometry of whole wounds from Col1a1-GFP reporter mice identified a CD3^-CD19^-Ly6G^-CD11b⁺F4/80⁺ resident macrophage population undergoing MMT from day 5 to 7 post-wounding (p<0.05). We identified the H3K36 methyltransferase Whsc1 was upregulated in wound macrophages in response to TGFβ, and siRNA knockdown of Whsc1 in bone marrow derived macrophages (BMDMs) decreased expression of Acta2, Col1a1, Col3a1 and H3K36me2 at fibrotic promoters (p<0.05). Myeloid-specific Whsc1 loss in Whsc1f^lox/flox;Lyz2^Cre+ mice impaired wound healing and decreased Acta2, Col1a1, and Col3a1 expression in wound macrophages. Local pharmacologic inhibition of Whsc1 in vivo disrupted wound healing (p<0.05). Flow cytometry of wounds isolated from Col1a1-GFP mice revealed fewer resident macrophages from mice on a diabetic diet (DIO) (2%) versus normal diet (15%) (p<0.05). Spatial sequencing of human wounds identified resident macrophages (FCGR1A, ITGAM, MERTK, CCR2, SIGLEC1, CX3CR1, CD163, LYVE1, MRC1, TIMD4, and CD9 positive) represented 4.4% of total macrophages (CD14, CD16, and CD68 positive), exhibited increased TGFB1 expression compared to non-resident macrophages, and were in closer proximity to fibroblast neighbors. scRNA-seq of human wounds revealed that Whsc1 expression was decreased in macrophages from diabetic wounds (p<0.05). In DIO BMDMs, Whsc1 was absent at fibrotic promoters after TGFβ treatment and remained at NFkB-regulated inflammatory gene promoters (Il1b, Il6, Tnf) bound to RelA. RelA knockdown in DIO BMDMs increased Whsc1 at fibrotic promoters and lead to increased fibrotic gene expression (p<0.05).

Conclusion: We show that MMT occurs in the resident macrophage subpopulation during wound healing and is regulated by TGFβ-Whsc1, which is disrupted in diabetic wound healing. These data identify the RelA-Whsc1 interaction as a novel target in diabetic wound treatment.