Proposal Summary Inflammatory bowel disease (IBD), comprised of ulcerative colitis and Crohn's disease, contributes to significant morbidity and mortality in the United States. The inflammation in IBD is initiated by a breakdown in the mucous layer and intestinal epithelial cells that act as a barrier in the gastrointestinal tract. Current therapeutics are inadequate in that they largely target systemic immunosuppression which have been associated with increased infection and malignancy risk as well as being efficacious in a minority of patients. These problems result in many IBD patients having active and debilitating disease. We have previously identified a key role for creatine, an energetic regulator, in intestinal epithelial cell barrier function and wound healing. This is significant in that IBD patients have decreased expression of the creatine transporter (CRT) which brings creatine into cells. More recently we have identified dysregulation of intestinal epithelial cell differentiation in cells that are deficient for CRT. Loss of creatine results in decreased goblet cell marker expression in CRT deficient organoids and decreased mucous containing goblet cells in the colon of CrtFloxVillinCre mice. This deficiency decreases mucous layer formation, which is expected as goblet cells are the major producers of the protective mucous layer which serves to protect the intestinal mucosa. In the follow proposal we aim to evaluate the hypothesis that the creatine pathway is integral to goblet cell function and dysregulation identified in CRT deficient epithelial cells will impact the microbiome and metabolism of the colonic lumen as well as contribute to increased colitis susceptibility. In Specific Aim 1, we propose to evaluate the contribution of glycolysis on goblet cell differentiation and activity in CRT deficient intestinal epithelial cells. We will also determine the cause of the dysregulation by evaluating key goblet cell signaling pathways. This aim is vital in that identifying the mechanism of goblet cell loss may provide options for therapeutic intervention. In Specific Aim 2 we will identify changes in microbiome that could be impacted by the decrease in mucous and other goblet cell produced factors as well as changes to the creatine content in the lumen. We will determine if these changes may contribute to a stressed metabolic colonic lumen environment. We believe that alteration to the microbiome may contribute to the inflammatory mucosal environment. Specific Aim 3 will assess for colitis disease susceptibility due to epithelial CRT loss in CrtFloxVillinCre mice. We will conduct salmonella colitis and TNBS colitis experiments which cause ulcerative colitis like inflammation in CrtFloxVillinCre mice and compare disease susceptibility. We will make use of findings in the prior aims to determine if there are interventions to prevent increased susceptibility in CrtFloxVillinCre mice including stimulation of differentiation pathways and...