Increased proliferation and reduced apoptosis of monocytes/macrophages are associated with their persistence in wounds of diabetic mice Jingbo Pang*, Mark Maienschein-Cline** and Timothy J. Koh*¶ *Center for Wound Healing and Tissue Regeneration, Department of Kinesiology and Nutrition, ** Research Informatics Core, University of Illinois at Chicago, Chicago, IL 60612 Monocytes and macrophages (Mo/MΦ) rapidly infiltrate into damaged tissue and play critical roles throughout the wound healing process. Non-healing diabetic wounds are associated with chronic inflammation and persistent accumulation of dysregulated Mo/MΦ. However, the mechanisms underlying the persistent accumulation of dysregulated Mo/MΦ in diabetic wounds remain poorly understood. The purpose of this study was to determine whether proliferation and/or apoptosis of wound Mo/MΦ contributes to their persistent accumulation in skin wounds in diabetic mice. First, cell cycle analysis by flow cytometry showed that the number and percentage of proliferating pro-inflammatory Ly6C+F4/80lo/- Mo/MΦ in the S/G2/M phases were higher in wounds of diabetic mice compared to non-diabetic mice. Moreover, unbiased scRNAseq analysis of flow sorted wound Mo/MΦ from non-diabetic and diabetic revealed a cluster, populated primarily by cells from diabetic wounds, for which genes associated with the cell cycle were enriched. With adoptive transfer of mixtures of CCR2-/- and CCR2+/+ Ly6Chi Mo to the wounds, we identified a critical role for CCL2/CCR2 signaling in promoting Mo/MΦ proliferation in the wound environment. Next, results from an Annexin V flow cytometry assay indicated significantly less Mo/MΦ apoptosis in wounds from diabetic mice, particularly in the pro-inflammatory Ly6C+ population. Furthermore, scRNAseq data implicated the TNF, MAPK, Jak-STAT, and FoxO signaling pathways in promoting wound Mo/MΦ apoptosis in wounds of non-diabetic mice while cell proliferation-related pathways appeared to be activated in wounds of diabetic mice. Taken together, these data demonstrate increased proliferation and reduced apoptosis of wound Mo/MΦ in diabetic mice, which may contribute to persistent Mo/MΦ accumulation and chronic inflammation in diabetic mice. These findings also highlight a novel role for the CCL2/CCR2 signaling pathway in promoting skin Mo/MΦ proliferation, as well as pathways that may regulate Mo/MΦ apoptosis during wound healing, which could be targeted to help resolve inflammation and improve healing.