Transcriptional Waves Induced by ATP and LPS in Primary Microglial Culture Reveal Multiple Inflammatory States

Abstract The primary role of microglia is to respond effectively to various disturbances to maintain brain homeostasis. In this study, we stimulated murine neonatal microglial cells with benzoyl ATP (bzATP) and lipopolysaccharide (LPS) and monitored their ability to secrete pro-inflammatory cytokines together with the changes in the transcriptome of the culture. Activation by bzATP, an agonist of purinergic receptors, induces a short-lived wave of transcriptional changes. However, a long-lasting transcriptional profile affecting thousands of genes occurs already at 3 hours after activation with a combination of bzATP and LPS. The transcriptomic profile is dominated by induced cytokines (e.g., IL1-a and IL1-b) and chemokines, in addition to upregulation of cytokine membrane receptors and their direct regulators. We identified the TNF and NF-kB signaling pathways as the principal hubs for many differentially expressed genes. Many of the reported inflammatory-related genes were upregulated by orders of magnitude. We conclude that the kinetics of microglial activation and specific combinations of stimuli yield coordinated and robust transcriptional waves. We propose that the primary microglia culture could be beneficial for studying brain inflammation associated with aging, injury, and neurodegenerative diseases.