PROJECT SUMMARY/ABSTRACT Non-healing wounds in patients with Type 2 Diabetes (T2D) are a major cause of increasing morbidity and mortality. Failure of wound healing in T2D patients represents the most common cause of amputation in the US with a 5-year mortality rate of nearly 50%. Thus, a critical need exists for understanding the wound healing defects in T2D in order to develop targeted therapies. We have utilized both genetic (db/db) and dietary (diet- induced obese) murine models of T2D as well as human wound tissue and blood samples collected from T2D patients to explore mechanisms of impaired wound healing. Our published and preliminary data demonstrate that interactions between wound macrophages (Mφs), and CD4+T cells, via Notch signaling, dictate CD4+T cell activation to ultimately regulate inflammation and healing in wounds. We and others have identified that a wound CD4+Treg cell phenotype is critical for normal tissue repair, while a TH17 phenotype promotes excess inflammation and impairs healing. Our preliminary data identify TLR4 signaling in wound Mφs upregulates the Notch ligand, DLL4, which then interacts with the Notch 1 and 2 receptors on CD4+T cells to promote TH17 as opposed to Treg differentiation. These interactions drive excess inflammation and pathologic healing in diabetes. Additionally, the DLL4-Notch pathway may be further upregulated in diabetic wounds via epigenetic mechanisms involving MLL1, a histone methyltransferase. MLL1 directly upregulates DLL4 in Mφs and can indirectly increase DLL4 via TLR4 signaling in Mφs. In addition, MLL1 can directly increase Notch 1 and 2 receptor expression in CD4+T cells, making them more receptive to Notch activation and promoting a prolonged TH17 response. These changes then dictate the pathologic TH17 phenotype in diabetic wounds. These results have led to our hypothesis 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. Further, increased downstream IL17A signaling increases epidermal and dermal cell mediated inflammation in diabetic tissue. Our data suggest that wound Mφ/CD4+T cell wound phenotypes may be restored via Mφ-targeted treatment (delivered locally to the wound via engineered nanoparticles) with MLL1 inhibitor(s) or local treatment with anti-DLL4 antibodies. To test our hypotheses, we will pursue the following aims: Aim 1: To examine the direct (MLL1) and indirect (TLR4/MyD88) regulation of DLL4 in wound macrophages during normal and diabetic conditions. Aim 2: To identify the role of Notch receptor activation on Treg/TH17 differentiation during normal and diabetic wound repair. Aim 3: To compare local and Mφ-targeted therapies to reduce Notch signaling, limit TH17 differentiation, improve healing and identify the cellular targets for IL17A signaling in wounds.