PROJECT SUMMARY Secondary complications of type 2 diabetes (T2D), including impaired wound healing, are increasing, and therapeutic options remain limited and only marginally effective. Failure of wound healing in T2D patients represents the most common cause of amputation in the US and has an a 5-year mortality rate of 50%. Thus, a critical need exists for understanding the pathophysiology of T2D woundsto identify novel mechanisms that can be therapeutically targeted. In normal wound healing, a coordinated immune response between macrophages (Mφs), and CD4+T cells is critical for a controlled inflammatory response and tissue repair. Although the molecular mechanisms that dictate these interactions following injury are not well understood, our group and others have identified that Notch signaling plays a critical role in wound healing. Depending on the specific ligand-receptor interactions, CD4+T cell activation results in differentiation into a specific T cell phenotype. It has been observed that CD4+T regulatory (Treg) cells are critical to ensure normal tissue repair, while TH17 phenotypes promote excess inflammation and impair healing; however, the mechanisms that control CD4+T cell activation in wound tissue are unknown. Our preliminary data show that increased Notch signaling between Mφs/CD4+T cells in diabetes can lead to increased TH17, decreased Tregs, excess inflammation and pathologic healing. While the precise regulation of Treg/TH17 differentiation during wound repair is unclear, our preliminary data identify that the Notch ligand, DLL4, is upregulated in diabetic wound Mφs, and that DLL4 interactions with the Notch 2 receptor on CD4+T cells promotes an inflammatory TH17 phenotype as opposed to Treg differentiation. We hypothesize that DLL4-mediated Notch receptor signaling in diabetic wounds polarizes CD4+T cells in the wound towards TH17 and promotes chronic inflammation and non-healing and that local blockade of DLL4 will decrease inflammation and improve healing in diabetic murine wounds. To test our hypotheses, we will pursue the following Specific Aims: 1) To identify the role of Notch 2 receptor activation on Treg/TH17 differentiation in acute and chronic diabetic murine wound models. 2) To determine if genetic or local therapeutic blockage of DLL4 ligand limits TH17 differentiation, decreases inflammation and improves diabetic wound repair.