P2Y 2 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 P2Y 2 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 P2Y 2 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 P2Y 2 targeting using either specific antagonists or siRNAs. P2Y 2 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 P2Y 2 KO mice secreted significantly less CXCL1/KC than WT controls. Altogether, extracellular nucleotide signalling regulates TLR3‐induced chemokine release in IECs through P2Y 2 receptors.