Abstract 145: Epigenetic Modifications of Bone-Marrow Progenitor Cells Promote an M1 Phenotype in Peripheral Macrophages in Diabetes

Abstract

Introduction: Diabetic wounds are characterized by a chronic inflammatory state that is maintained by overexpression of pro-inflammatory cytokines generated by immune cells, namely macrophages. To understand this imbalance, it is critical of understand how changes in immune cells at the systemic level influence the peripheral immune cells. Work from our lab has shown that cytokines induce a histone post-translational “epigenetic signature” at promoter sites of specific genes. However, the role of histone modification of DNA on diabetic progenitor and differentiated immune cells and their impact on chronic inflammation in the periphery has not been studied. We hypothesized that epigenetic changes to bone marrow cells influences peripheral macrophage phenotypes. Methods: Bone marrow was harvested from 7 diet-induced obese mice (DIO) (mean glucose= 290) and 7 wildtype (WT) controls. In some of the mice, lin -/ckit+ population was isolated and flow cytometry (FACS) analysis was performed to isolate hematopoietic stem cells (HSC) and myeloid progenitor cells (MYP). HSC were defined as cells expressing sca1+/CD150+/flt3+ and MYP co-expression of sca1+/CD150+/FcgR-/CD105low. HSC and MYP were analyzed via FACS for the presence of chromatin marks. In other mice, BMD macrophages were isolated and propagated in cell culture. Macrophages were either untreated or skewed towards an M1 or M2 phenotype with IF-gamma or IL-4, respectively. Levels of protein and mRNA of M1/M2 products were analyzed. Additionally, at 6, 24 and 72 hours the macrophages were assessed for expression of chromatin remodeling marks via FACS analysis. Results: HSC had a 2 fold decrease in the expression of H3K9 histone methylation marks in the DIO mice as compared to WT. (Figure 1A) This pattern was also observed in the more differentiated MYP cells. (Fig 1B) Macrophages isolated from diabetic mice also reflected these histone changes, supporting the role of histone methylation on fully-differentiated macrophage gene expression. (Fig 1C) DIO macrophages demonstrated increased levels of TNF-alpha and CCL3, classic M1 phenotype markers. (Fig 1D) Conclusions: Epigenetic changes initiated in the bone marrow progenitor cells effect immune cells in the periphery, and may contribute to the chronic inflammation seen in diabetics. Manipulation of these histone modifications at the bone marrow level could allow for the development of therapies to prevent chronic inflammation in the periphery of diabetics.