Purinergic Signaling in Intestinal Inflammation - PROJECT SUMMARY Purines are important signaling molecules that influence multiple pathways in the human body by activating purinergic receptors on target cells. Recent research has identified that purines and their corresponding purinergic receptors play critical roles in infectious disease. However, much of this research has focused on purinergic signaling within immune cells and the enteric nervous system, leaving the role of epithelial cells in the gastrointestinal tract largely unexplored. Our group has identified that the purinergic receptor P2Y1 is highly expressed in the human intestine and in human intestinal organoids. We found that activation of P2Y1 stimulates robust calcium signaling and cell-cell communication; pathways known to be involved in epithelial responses to injury. We generated CRISPR-Cas9 knockout (KO) organoids of P2Y1 and found that these cells had reduced calcium signaling and decreased wound closure rate. We also generated epithelial specific P2Y1 KO (VillinCre:P2Y1flox/flox) mice and found that these P2Y1 KO animals were more susceptible to dextran sodium sulfate (DSS) induced colitis. Both our organoid and animal models suggest that P2Y1 is critical for proper intestinal wound healing and response to injury. Finally, we found Ulcerative Colitis patients with active inflammation have a significant reduction in P2Y1 expression, but P2Y1 expression returns to normal when grown as organoids and removed from the inflammatory environment. These data suggest that P2Y1 is influenced by inflammatory signals and maybe a target for promoting wound repair in the inflamed gut. Our central hypothesis is that epithelial purinergic P2Y1 signaling is involved in intestinal inflammation during colitis and plays an important role in repair. The objective of this research is to elucidate the role of epithelial purinergic signaling in intestinal inflammation and the mechanistic consequences of these pathways. Aim 1 will delineate the role of epithelial P2Y1 purinergic signaling in vivo during acute and chronic colitis and wound biopsy punch. We anticipate that deficiency of P2Y1 (Villincre:P2Y1flox/flox mice) will result in worsened DSS and T-cell transfer induced colitis and delayed healing compared to WT mice. In Aim 2 we will identify the mechanism by which P2Y1 signaling drives wound healing and determine how inflammatory signals affect P2Y1 signaling pathways using human colonic organoids derived from healthy and ulcerative colitis patients. We anticipate P2Y1 activates both proliferative (ERK1/2) and cell migration (Itpkb) pathways to promote proper repair following injury. However, in the presence of pro-inflammatory stimuli, we postulate that P2Y1 expression and its downstream signaling pathways will be downregulated. This study will provide evidence for the importance of P2Y1 purinergic signaling in epithelial cells in the context of intestinal inflammation during colitis. Collectively, these experiments will advance our understanding of intestinal inflammation and may unveil potential novel therapeutic targets.
