Extracellular Nucleotide Signaling Modulates NOD2 Agonist-Induced CXCL8 Secretion in Human Monocytes

NOD2 sensing of muramyl dipeptide (MDP) promotes the production of chemokines by monocytes, which contribute to innate immune cell recruitment. Increasing evidence indicates that extracellular nucleotides shape innate immune outputs through P2 receptors. In this study, we demonstrate that P2 receptor signaling regulates NOD2-agonist-driven CXCL8 secretion in the human monocytic THP-1 cell line and in human primary monocytes and also contributes to NOD2-agonist-driven CCL2 secretion in THP-1 cells. Stimulation with bioactive MDP induced a robust, concentration-dependent increase in CXCL8 secretion, whereas the inactive MDP stereochemical control peptide, MDPc, had no effect. Limiting extracellular nucleotide availability with apyrase markedly reduced MDP-induced CXCL8 release, and broad P2 receptor antagonism produced a comparable inhibition, indicating that extracellular nucleotide signaling supports NOD2-agonist-driven output. Supplementation with exogenous nucleotides further enhanced CXCL8 secretion in MDP-stimulated cells. Receptor profiling using selective antagonists identified P2Y2 in THP-1 cells and P2Y6 in human primary monocytes as the dominant contributors to this amplification, as pharmacological inhibition of P2Y2 and P2Y6, respectively, strongly suppressed CXCL8 secretion, whereas blockade of P2Y1 had no effect. In agreement with these findings, selective activation of P2Y2 in THP-1 cells induced CXCL8 secretion and potentiated MDP-induced CXCL8 release. In addition, P2Y2 signaling regulated NOD2-agonist-mediated CCL2 secretion in THP-1 cells. Together, these findings demonstrate that extracellular nucleotide signaling stimulates CXCL8 secretion and amplifies NOD2-agonist-induced CXCL8 secretion in monocytic cells, with additional regulation of CCL2 observed in THP-1 cells.