(WHS-I.01) SINGLE-CELL TRANSCRIPTOMICS REVEALS REDUCTION OF A POPULATION OF MONOCYTE-DERIVED DENDRITIC CELLS ASSOCIATED WITH IMPAIRED HEALING IN DIABETIC WOUNDS OF MICE
Wednesday, May 15, 2024
1:45 PM – 4:00 PM East Coast USA Time
Diabetes induces dysregulation of a spectrum of inflammatory cells which contributes to persistent inflammation and defective tissue repair responses. To explore dysregulation of subsets of monocyte/ macrophage lineage, we isolated and pooled Live CD45+CD11b+Ly6G- cells from excisional skin wounds of non-diabetic C57Bl/6 and diabetic db/db mice on day 3, 6, and 10 post-injury and performed scRNAseq analysis using the 10x Chromium platform. Cells from non-diabetic vs diabetic wounds exhibited strikingly different phenotypes throughout the healing process. Although wound cells in non-diabetic mice showed an expected transition from pro-inflammatory to pro-healing phenotypes, cells from diabetic mice did not show this transition. Interestingly, a cluster expressing high levels of Cd209a, Cd74 and other genes associated with antigen presenting cells (APC) was populated primarily by cells from non-diabetic mice with significantly lower contribution from diabetic mice. These data indicate decreased accumulation of APCs in wounds of diabetic mice and were confirmed by flow cytometry analysis using markers for this APC cluster. To determine the origin of these cells, we examined this APC population in skin wounds from CCR2 knock-out (KO) mice which lack circulating monocytes. Accumulation of these APCs was almost completely ablated in wounds of CCR2 KO mice compared to their wild-type counterparts, suggesting these cells originate from circulating monocytes. Importantly, adoptive transfer of bone marrow naïve Ly6C+ monocytes to skin wounds indicated that donor cells gained dendritic cell markers including MHC II, CD11c, CD74 and CD209a after 20-hour incubation in the wound environment, confirming the monocyte origin of these APCs. Finally, to begin to understand the regulation of this APC population, we used the BITFAM model to infer TF activity in these cells during skin wound healing, incorporating both scRNAseq and scATACseq data. Predicted TF activity associated with this APC population included PRDM1, IRF4 and STAT4 activity, which was supported by significantly higher protein levels in these cells compared to other wound monocyte and macrophage populations. Previous studies have demonstrated monocytes have the capacity to differentiate into APCs called monocyte-derived dendritic cells which can adopt multi-functional capabilities during inflammation. We are currently performing experiments to elucidate the function of these cells during wound healing. Together, our data demonstrated the significant reduction of monocyte-derived APC in skin wounds of diabetic mice, which may contribute to impaired healing in these mice.