(PC210) The TGFβ-Whsc1 axis drives macrophage to myofibroblast transition during wound repair

Objectives: During the proliferative phase of tissue repair macrophages can transition to myofibroblasts, which promote wound contraction and closure. The mechanisms that control this transition remain unknown and are not well studied. Thus, the objectives of this study were to examine the epigenetic mechanisms controlling macrophage to myofibroblast transition (MMT) and how this process may be disrupted in diabetic wound healing.

Methods: Here, using a combination of murine transgenic models of Whsc1 deletion (Whsc1flox/floxLyz2Cre) and disease, loss of function approaches, pharmacologic inhibition in vivo, and single cell RNA-seq (scRNA-seq) in human wounds, we identified that a histone methyltransferase, Whsc1, regulates the fibrotic gene program in macrophages during wound repair. Additionally, we use spatial sequencing of human wounds and flow cytometry with a murine genetic reporter to identify that MMT occurs in a select resident macrophage population.

Results: We identified that Whsc1 was upregulated in wound macrophages (CD3-CD19-NK1.1-Ly6G-CD11b+) in response to TGFβ. Genetic loss of Whsc1 in wound macrophages from Whsc1flox/floxLyz2Cre mice as well as siRNA knockdown of Whsc1 in bone marrow derived macrophages (BMDMs) decreased TGFβ-dependent expression of myofibroblast markers, Acta2, Col1a1, Col3a1. Importantly, local pharmacologic inhibition of Whsc1 in vivo significantly impaired wound healing. We found wound healing was also delayed in mice with genetic deletion of Whsc1, suggesting that Whsc1 is critical for normal wound repair. Mechanistically, we demonstrate that Whsc1 mediates TGFβ-mediated H3K36 dimethylation at fibrotic gene promoters in wound macrophages and this is important for MMT. Using spatial sequencing of human wounds and flow cytometry, we also show that this transition occurs in a select population of resident macrophages and is disrupted in diabetic wound healing. Specifically, Whsc1 expression was decreased in human macrophages analyzed by scSeq of diabetic wounds and in murine wound macrophages from a diabetic model (DIO). Interestingly, in DIO BMDMs, Whsc1 did not traffic to fibrotic promoters after TGFβ treatment, but rather remained at NFkb-regulated inflammatory gene promoters (Il1b, Il6, Tnf). On further examination of this, using co-immunoprecipitation and proximity ligation assay in BMDMs, we demonstrated increased physical interaction between RelA and Whsc1 in diabetic macrophages. Knockdown of RelA in DIO macrophages increased Whsc1 enrichment at fibrotic promoters, suggesting a potential therapeutic strategy.

Conclusions: The identification of TGFβ-Whsc1 as a driver of macrophage-myofibroblast transition in normal and pathological tissues allows for development of novel therapies to improve tissue repair.