Work

23. Single Cell Sequencing Reveals Androgens Drive Monocyte-lineage Cell Clusters Towards Pro-inflammatory And Pro-angiogenic Transcriptomic States

Year: 2024

Type: article

Abstract: Purpose: The molecular mechanisms underlying androgen-driven aberrant wound healing (WH) remain unexplored. Our previous human and animal studies have consistently demonstrated a significant correlation between testosterone and abnormal WH. Given the complexity, a comprehensive understanding of interactions between sex hormones and wound environment requires high-resolution analysis. We performed single-cell RNA sequencing to obtain insight into key immune cell populations and molecular pathways influenced by testosterone (T). Methods: XX and XY Sprague-Dawley rats underwent surgical castration, with half receiving bi-weekly testosterone injections (+T). All rats received bilateral 15mm dorsal excisional wounds two weeks post-gonadectomy. Wounds were harvested on postoperative day 10 (N=8; 4+T/4-T) and subjected to mechanical and enzymatic digestion, yielding single-cell suspensions. Oligo-labeling was performed using 10X Genomics CellPlex Multiplexing System. Single-cell RNA-sequencing, QA/QC, and barcode resolution were performed by the Whitehead Institute Genome Technology Core. Analysis was conducted using R with Seurat and DESeq2. Results: Eight wound samples weighing 0.6-1.38 g were processed with weight correlating to single-cell suspension counts (r=0.85, 87-98% viability). We analyzed 6,514 cells by scRNA-seq. Initial clustering revealed 11 distinct cell clusters including monocytes and macrophages (Mø), T cell lymphocytes, granulocytes, endothelial cells, and fibroblast. We further characterized the Mø phenotypic shifts in clusters 2 & 3, distinguished by classical markers of Cd14, Cd163, and CD68. Through gene set enrichment analysis (GSEA), +T Mø displayed upregulation of TNF-α signaling via NFK-β, and pI3K/Akt/mTOR signaling pathways. Heatmaps based on gene set lists showed a mixture of pro-inflammatory immune response and pro-angiogenic signatures, namely Il-1a, Tnf-a, Vegf-a, Cd44, Cd83, Il-7r, Rei1, and Traf-1 in +T. Volcano plot visualization of differentially expressed genes (DEGs) in Mø clusters showed statistically significant changes in Cxcl-3, Cd86, Cd83, and Ctsf, associated with increased phagocytic activity, antigen presentation, and co-stimulation of T cell lymphocytes. Conclusions: Single-cell analysis offers a dynamic view of the cellular landscape during wound healing in the presence of circulating testosterone. Highly enriched genes related to NFKβ and pI3K/Akt/mTOR signaling pathways in Mø population led to increased pro-inflammatory signatures at POD10, noted by upregulation of Tnf and Il-1a. Importantly, testosterone also increased pro-angiogenic phenotypes of Mø, with upregulation of Vegfa and Cd44, suggesting a possible mechanism for hypertrophic scarring. Ongoing work is characterizing T-altered wound immune cell crosstalk. Enrichment of NFKβ signaling hub adaptor proteins such as Rei1 and Traf1 may open future directions targeting canonical and non-canonical pathways of NFkB to combat the harmful effects of T in the wound milieu.

Source: Plastic & Reconstructive Surgery Global Open

DOI: 10.1097/01.gox.0001015184.76810.e5