W. J. Melvin1, F. M. Davis1, C. O. Audu1, E. Barrett1, K. Mangum1, S. Wolf1, A. Joshi1, J. Shadiow1, S. B. Sharma1, R. Kai1, A. Obi1, B. B. Moore1, K. Gallagher1 1University Of Michigan, Ann Arbor, MI, USA
Introduction: Non-healing wounds in patients with Type 2 Diabetes (T2D) are a major cause of increasing morbidity and mortality. We and others have shown that a prolonged pro-inflammatory state is typical of pathologic diabetic wound healing. Regulatory T cells (Treg) are a subset of CD4+ cells that are known to be critical for physiologic wound healing and transitioning the wound microenvironment to a reparative state from an initial inflammatory state. Given that Notch signaling is known to regulate T cell phenotype in a context-dependent manner in other settings, we hypothesized that it regulates T cell phenotype in wound healing, and that decreased activation of Notch signaling in diabetic wounds leads to decreased Treg phenotype and subsequent pathologic inflammation.
Methods: Human single cell transcriptional profiling of diabetic wounds versus control wounds was obtained from amputated specimens (n=3/group) and analyzed for presence of Tregs. Next, using a murine wound healing model (4mm punch biopsy), wound T cell phenotypes were analyzed by flow cytometry from C57BL/6 diet-induced obese (DIO) mice (a model of T2D) at day 3, 5, and 7 post-wounding (n=5/group). Wound CD4+ T cells were isolated using magnetic cell sorting and analyzed for Notch signaling expression via quantitative PCR (qPCR). C57BL/6 mice with CD4+-specific deficiency in Notch signaling (DNMAML-CD4cre+) were wounded and compared to control (cre-) littermates (n=5/group) for wound healing via daily photo analysis and presence of Tregs via flow cytometry. Wound healing curves were analyzed using Image J software. Statistical significance was determined using Student’s t-test.
Results: Human single cell transcriptional profiling of diabetic wounds versus control wounds revealed decreased Tregs in diabetic wounds. Diabetic mice had less wound Tregs (CD4+CD25+FOXp3+) compared to nondiabetic control mice, and decreased expression of Notch target genes Hey1 and Hes1, indicating decreased Notch signaling. Similarly, DIO DNMAML-CD4cre+ had less wound Tregs compared to cre- littermate controls, and also impaired wound healing (Fig 1).
Conclusion: Tregs are decreased in diabetic wounds, driving persistent uncoordinated inflammation and pro-inflammatory phenotypes in other cell types. Notch signaling is decreased in diabetic wound T cells, decreasing Tregs and leading to impaired wound healing. Mice with T cell-specific Notch signaling deficiency also demonstrate decreased Treg formation and impaired wound healing. Thus Notch signaling may offer a potential therapeutic target to reduce pathologic inflammation in diabetic wounds.
