PEOPLE WITH ULCERATIVE COLITIS HAVE ALTERED EPITHELIAL PURINERGIC P2 RECEPTOR EXPRESSION AND FUNCTION

Abstract BACKGROUND Purines are well-established as potent signaling molecules that regulate cellular homeostasis through activation of purinergic receptors. There are 19 distinct purinergic receptor subtypes, classified as P1 and P2 receptors. While purinergic receptors are known to play important roles in immune cell responses during inflammation and infection, their function in the intestinal epithelium, especially in inflammatory bowel disease (IBD), remains largely unknown. We hypothesized that key purinergic receptors are altered during IBD. Methods & Results We queried single cell RNA sequencing data from the Human Protein Atlas and CZ CELLxGENE Discover databases to assess purinergic receptor distribution in healthy human colon. In silico analysis revealed limited P1 receptor expression in the intestinal epithelium but robust expression in immune cell types. By contrast, healthy small and large intestine epithelium expressed the P2 receptors P2RY1, P2RY2, and P2RX4 across nearly all epithelial cell types, while immune cells were enriched for distinct P2X and P2Y receptors. To address whether these receptors were altered in IBD, we examined RNAseq data from 206 new-onset people with ulcerative colitis and control non-IBD patients during diagnostic colonoscopy (GSE109142). Ulcerative colitis patients were stratified by histological severity and levels of fecal calprotectin. We found that epithelial associated P2RY1, P2RY2 and P2RX4 were all significantly reduced in ulcerative colitis patients with increasing levels of inflammation. Interestingly, we did not observe any changes in P1 receptor expression. In contrast to the epithelial-associated purinergic receptors, we observed increased abundance of several immune-associated purinergic receptors, including P2RY6, P2RY13, P2RY14, P2RX1, P2RX5, and P2RX7. To evaluate the functional consequences of decreased P2 receptor expression, we used human intestinal organoids expressing the calcium indicator GCaMP6s. Inhibition or knockout of P2Y1 significantly reduced basal calcium signaling, indicating impaired epithelial communication. Furthermore, blocking P2Y1, but not P2Y2, delayed wound healing in epithelial scratch assays, suggesting a role in epithelial injury repair. CONCLUSIONS These findings suggest an imbalance in epithelial and immune cell purinergic signaling in the setting of ulcerative colitis and point to unique roles of purinergic signaling with the intestine.