3375 Chronic inflammation promotes intestinal macrophages to become modulators of the Notch pathway

Abstract

OBJECTIVES/SPECIFIC AIMS: The purpose of this research was to investigate how chronic inflammation promotes the generation of proinflammatory intestinal macrophages and if macrophages contribute to intestinal inflammation through Notch activation. METHODS/STUDY POPULATION: We utilized two animal models of chronic colitis, the chronic DSS-induced colitis mouse model and the spontaneous enterocolitis development in IL-10-deficient mice to investigate the role of chronic inflammation in the generation of proinflammatory intestinal macrophages and its influence in notch signaling. Bone marrow-derived monocytes were collected from each group and differentiated into macrophages (BMM) for gene and protein analysis. Ex vivo phenotypical and functional analysis of colonic macrophages was assessed as was the presence of goblet cells and mucosal T cells. In addition, we analyzed the development of goblet cell differentiation in colonoids in a co-culture system with proinflammatory macrophages. RESULTS/ANTICIPATED RESULTS: Our chronic inflammation models revealed an increase in proinflammatory macrophages present in the lamina propria and that these cells expressed significantly higher levels of notch ligand, Jagged1. Jagged 1 has been shown to enhance TH1 differentiation and T cells isolated from the mucosa of both chronic colitis models display strong TH1 skewing compared to controls. Chronic inflammation also contributes to intestinal barrier defects, enhanced permeability and bacterial translocation. We believe this enhanced intestinal permeability and subsequent bacterial translocation promote Jagged1 expression in intestinal macrophages. To support this concept, we show TLR stimulation induces the upregulation of Jagged1 in BMM. Additionally, the generation of BMM from our chronic DSS-induced colitis mice or age matched controls, revealed BMM derived from a host of chronic inflammation were skewed to a proinflammatory state prior to stimulation showing increased gene expression of several proinflammatory molecules including IL-1α, IL-1β, IL-12 and TNF-α. This would suggest monocytes migrating to the intestinal mucosa have more potential to become proinflammatory instead of traditional anti-inflammatory macrophages. Furthermore, proinflammatory notch ligand-positive macrophages co-cultured with colonoids, derived from unperturbed mice, significantly decreased the number of mucus producing goblet cells. In support of this observation, notch activation in intestinal stem cells promote absorptive (i.e. colonocytes) cell differentiation and prevents secretory cell (i.e. goblet cells) differentiation. DISCUSSION/SIGNIFICANCE OF IMPACT: Taken together, our results strongly suggest chronic inflammation modulates macrophages role in maintaining intestinal homeostasis through possible notch activation in both T cells and the intestinal epithelial barrier.