Cholinergic agonist PNU282987 induces pro-inflammatory gene expression via the MCOLN1-TFEB pathway in murine macrophages

Neurotransmitter signaling in macrophages is a modulatory mechanism during inflammation. Treatment of macrophages with acetylcholine, or agonistic molecules carbachol and PNU282987, considered to be specific for the Alpha7 nicotinic ACh receptor (α7NAChR), represses cytokine expression in inflammatory disease models, leading to the idea that α7NAChR stimulation could modulate inflammation in health and disease. However, their effects on resting macrophages are largely unaddressed. This study assesses the impact of PNU282987 on macrophage phenotypes in vitro , focusing on bone marrow-derived macrophages (BMDMs) and RAW264.7 cells. We found that PNU282987 induces a specific pro-inflammatory gene expression profile, elevating Ifnb1, Il1b, Il6, and Tnf levels. However, this did not translate to increased cytokine secretion. Unexpectedly, both BMDM and RAW264.7 cells showed no detectable α7 nicotinic acetylcholine receptor expression, challenging the proposed action mechanism of PNU282987. Instead, our data suggest a non-receptor-mediated pathway, where PNU282987 may activate the stress-responsive TFEB transcription factor via ROS generation, implicating the MCOLN1-calcineurin pathway. Our findings reveal that the primary action of PNU282987 in resting macrophages is through non-receptor-mediated pro-inflammatory gene expression and TFEB activation, offering new insights into its pharmacological effects beyond the conventional receptor-based mechanisms.