Transcription Factor Activity Regulating Macrophage Heterogeneity during Skin Wound Healing

Abstract Monocytes and macrophages (Mos/Mϕs) play diverse roles in wound healing by adopting a spectrum of functional phenotypes; however, the regulation of such heterogeneity remains poorly defined. We enhanced our previously published Bayesian inference TF activity model, incorporating both single-cell RNA sequencing and single-cell ATAC sequencing data to infer transcription factor (TF) activity in Mos/Mϕs during skin wound healing. We found that wound Mos/Mϕs clustered into early-stage Mos/Mϕs, late-stage Mϕs, and APCs, and that each cluster showed differential chromatin accessibility and differential predicted TF activity that did not always correlate with mRNA or protein expression. Network analysis revealed two highly connected large communities involving a total of 19 TFs, highlighting TF cooperation in regulating wound Mos/Mϕs. This analysis also revealed a small community populated by NR4A1 and NFKB1, supporting a proinflammatory link between these TFs. Importantly, we validated a proinflammatory role for NR4A1 activity during wound healing, showing that Nr4a1 knockout mice exhibit decreased inflammatory gene expression in early-stage wound Mos/Mϕs, along with delayed wound re-epithelialization and impaired granulation tissue formation. In summary, our study provides insight into TF activity that regulates Mo/Mϕ heterogeneity during wound healing and provides a rational basis for targeting Mo/Mϕ TF networks to alter phenotypes and improve healing.