The intestinal epithelium is vital for maintaining immune homeostasis. The maintenance of homeostasis requires the host immune system to be able to differentiate between healthy, homeostatic tissue and commensal microorganisms, and danger from pathogens or host tissue damage. Damaged or dying cells release endogenous molecules termed alarmins, such as ATP. ATP is a ligand for the members of the P2 family of purinergic receptors. P2X and P2Y receptors are widely expressed in immune and non-immune cell types in humans and mice, and mediate a range of normal functions, as well as inflammatory responses. However, the function of these receptors differs between macrophages and intestinal epithelial cells, and relatively little is known about ATP sensing in gut epithelium and published studies often conflict. This thesis sought to characterise mouse intestinal epithelial cell expression of the P2 family of receptors, and the downstream function of ATP signalling in these cells. The first aim investigated ATP sensitivity in epithelial cells and expression of P2X receptors. In contrast to macrophages, IECs did not upregulate Nlrp3, Il18 or Il1Î² in response to LPS priming and were not susceptible to P2X7-mediated cell death. P2X7 expression was not found in IECs, and while P2X4 was expressed, it likely did not contribute to ATP-induced calcium mobilization. However, an antagonist of activity downstream of P2Y suggested that ATP-induced calcium mobilization may be mediated by the P2Y family of receptors. The second aim and results chapter further charted purinergic receptor expression and focused on P2Y in IECs. IECs expressed multiple P2Y receptors, but most prominently the ATP and UTP receptor, P2Y2. Calcium mobilization assays with antagonists suggested that P2Y2 may be, at least in part, responsible for IEC responses to ATP. The final aim then sought to assess the outcome of ATP, ADP and UTP signalling in IEC. ATP stimulation significantly contributed to LPS-induced transcription of Il6 mRNA. In addition, ATP and ADP induced significant upregulation in the transcription of the chemokines Cxcl1 and Ccl2. LPS-primed IECs, stimulated with ATP, ADP and UTP, showed significantly upregulated Cxcl1, Cxcl2, Ccl2, and Cxcl10 transcription compared to control cells, although nucleotide treatment of LPS-primed cells did not significantly increase transcription compared to LPS priming alone. While LPS did induce significant increases in CXCL2 protein secretion, significant increases were not observed in CXCL1, IL6, or CCL5 in a short-term (2-hour) protein assay. ATP signalling has been linked to cell proliferation and was also shown to increase IEC proliferation in a dose-dependent manner. Taken together, these findings suggest that P2Y receptors are involved in mediating intestinal epithelial responses to ATP signalling, leading to the upregulation of pro-inflammatory cytokines and proliferation. The results in this thesis suggest that intestinal epithelial cells respond to ATP detection by promoting immune cell recruitment to sites of damage and injury, whilst promoting epithelial renewal and repair.
- Intestinal Epithelial cells
- Purinergic receptors