P2Y₂ Receptor Controls TLR3-Induced Chemokine Production in Intestinal Epithelial Cells

Toll-like receptors (TLRs) and P2 receptors are key regulators of innate immunity. During infection, pathogen-associated molecular patterns (PAMPs) activate TLRs, whereas extracellular nucleotides engage P2 receptors and influence inflammatory responses. Here, we show that polyinosinic:polycytidylic acid (poly[I:C])-activated TLR3 and P2Y₂ signalling functionally interact in intestinal epithelial cells (IECs) to regulate chemokine production. HT-29 cells were stimulated with PAMPs, including the TLR3 agonist poly(I:C), in the presence or absence of P2 receptor signalling inhibitors. CXCL8/IL-8 or CXCL10/IP-10 secretion was assessed by ELISA and mRNA expression by quantitative real-time PCR (RT-qPCR). Primary IECs from P2Y₂ knock-out (KO) and wild-type (WT) mice were also treated with poly(I:C), and CXCL1/KC secretion was measured. Poly(I:C) stimulation induced robust CXCL8/IL-8 and CXCL10/IP-10 release in HT-29 cells. This response was inhibited by nucleotide scavenging, P2 receptor blockade, and P2Y₂ targeting using either specific antagonists or siRNAs. P2Y₂ was the dominant receptor expressed, and its ligands ATP/UTP constitutively released by these cells amplified CXCL8/IL-8 production induced by a suboptimal concentration of poly(I:C), while alone they had no effect. In support of these findings, primary IECs from P2Y₂ KO mice secreted significantly less CXCL1/KC than WT controls. Altogether, extracellular nucleotide signalling regulates TLR3-induced chemokine release in IECs through P2Y₂ receptors.