Mixed-Lineage Leukemia 1 (Mll1) Drives Notch Signaling and Regulates T-Cell Phenotype and Inflammation in Diabetic Wounds

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 dampened local initial inflammatory response, followed by a prolonged pro-inflammatory state, is typical of diabetic wound healing. Previous literature has shown Notch signaling drives naïve CD4+ differentiation into anti-inflammatory Tregs. We hypothesized that loss of Notch signaling via MLL1, an epigenetic enzyme altered in diabetes, leads to loss of diabetic wound Tregs and pathologic inflammation. METHODS: Murine wound T cell phenotypes were analyzed by flow cytometry from C57BL/6 diet-induced obese (DIO) mice (a model of T2D) at multiple timepoints. C57BL/6 mice with CD4+-specific deficiency in Notch signaling (Notch1CD4cre+, Notch2CD4cre+, DNMAML-CD4cre+), and CD4+-specific deficiency of MLL1 (MLL1CD4cre+) were wounded and compared to control (cre-) littermates. T cell RNA expression was analyzed using quantitative PCR (qPCR). RESULTS: Notch1 and Notch2 receptor and Notch intracellular cleaved domain (NICD) was decreased in diabetic wound T cells compared to nondiabetic CD4+ cells. We then found there was a decrease in MLL1 activity in diabetic T cells. Furthermore, T cells from wounded MLLf/fCD4cre+ mice demonstrated decreased NICD, Notch gene expression and wound Tregs. Furthermore, multiple strains of mice lacking intact T cell Notch signaling all demonstrated loss of wound Tregs and increased wound inflammation via IL-17. CONCLUSION: In summary, in diabetic wound CD4+ cells, a decrease of MLL1 regulates Notch signaling and subsequently decreases Tregs, contributing to pathologic prolonged inflammation and impaired healing in diabetic wounds.