P2X4 purinergic receptor loss alters macrophage homeostasis, cytokine secretion, and phagocytosis in a myeloid-specific P2X4 purinergic receptor knockout mouse.

INTRODUCTION: The P2X4 purinergic receptor is robustly expressed on tumor-associated macrophages; however, the role of P2X4 purinergic receptors on macrophages is not fully defined. To investigate the functional role of P2X4 purinergic receptor in macrophages, we generated and characterized a myeloid-specific P2X4 purinergic receptor knockout (P2X4ΔM) mouse. METHODS: Immune profiles were assessed across multiple tissues using multiplex immunohistochemistry and flow cytometry. Bulk RNA sequencing, gene set enrichment analysis (GSEA), and Gene Ontology over-representation analysis (ORA) were performed to determine the transcriptional consequence of P2X4 purinergic receptor loss in macrophages. Array and ELISA analyses of cytokine secretion, phagocytosis assays and Seahorse extracellular flux analysis of oxygen consumption were used to assess the functional role of P2X4 purinergic receptors in bone marrow-derived macrophages. RESULTS: P2X4 purinergic receptor loss in macrophages from male mice resulted in reduced tissue-resident macrophage density in liver, spleen, and peritoneal tissues. In addition, male P2X4ΔM mice had increased B cell populations among peritoneal cells. Bulk RNA sequencing analysis identified P2X4 purinergic receptor as the predominant P2 purinergic receptor expressed in murine macrophages and demonstrated that its deletion did not appear to induce compensatory expression changes in other P2 purinergic receptors. GSEA and Gene Ontology ORA determined an enrichment of interferon-β response and angiogenesis-related pathways in macrophages from male mice. Mitochondrial and metabolic pathways were enriched in macrophages from female mice. P2X4 purinergic receptor deficient macrophages exhibited reduced expression of pro-angiogenic Lysyl Oxidase-Like 2 whereas anti-angiogenic gene Plexin-D1 expression was increased. Functionally, P2X4 purinergic receptor deletion attenuated LPS-induced IL-6 and MCP-1 secretion in bone marrow derived macrophages and P2X4ΔM macrophages exhibited increased phagocytic activity. CONCLUSIONS: Collectively, these findings demonstrate that macrophage specific P2X4 purinergic receptor signaling regulates macrophage homeostasis, cytokine secretion, phagocytosis, and alters the expression of angiogenesis-associated genes.