Single Cell RNA-Seq Analyses of Healthy Lower Extremity Skin and Diabetic Foot Ulcers Uncover Distinct Immune Landscape of Diabetic Wound Healing

Fifteen percent of diabetes mellitus (DM) patients are expected to develop a diabetic foot ulcer (DFU) within their lifetime. DFUs represent an ever increasing burden for the healthcare system. Chronic wounds, lack the linear progression from one classical phase of healing to the next and are mainly characterized by the persistence of the inflammatory phase. To define the transcriptome landscape of healthy uninjured skin, DM and DFU skin, we performed transcriptome profiling on skin specimens from foot surgeries obtained from 4 controls, 4 DM patients without DFU and on samples from 2 DM patients after DFU debridement . We generated the single cell transcriptome profiles of 3,766 and 3,469 cells from non-DM and DM patients and of 660 cells from patients with active DFUs. Further, bioinformatics analysis identified clusters of cells with similar transcriptome states. The most abundant cell type was fibroblast. Other clusters included adipocytes and endothelial cells. T-cells, monocytes, and macrophages were the main immune cell types identified. As macrophages are critical for healing of DFUs, we estimated the abundance of total macrophages and their polarization. Approximately 10% of cells were macrophages in each group while DFU group had significantly more M1 polarized as compared to M2 (M1/M2=2.4 times). Supervised analysis identified a set of 66 and 1genes that are significantly differentially expressed in macrophages of DFU and DM patients respectively as compared to controls. A master regulator (MR) analysis in DFU patients identified activation of inflammatory and angiogenesis related MRs (e.g., STAT3, IL15). while in DM patients it identified activation of distinct inflammatory MRs (e.g., SMAD3, CD44). In summary, we present for the first time sc-RNAseq data on lower extremity skin samples that could help delineate the molecular mechanisms of dysregulated wound healing and identify novel therapeutic targets.