Identification of the genes regulated by the 5-HT2B serotonin receptor agonist BW723C86 in human M-CSF-primed monocyte-derived macrophages

Peripheral serotonin (5-HT) exacerbates or limits inflammatory pathologies (pulmonary arterial hypertension, cardiac valve degeneration, systemic sclerosis, gut disorders, neuroendocrine neoplasms, arthritis) through interaction with seven types of 5-HT receptors (5-HT1-7). As central regulators of inflammation, macrophages are critical targets of 5-HT, which promotes their anti-inflammatory and pro-fibrotic polarization primarily via the 5-HT7-Protein Kinase A (PKA) axis. However, anti-inflammatory human macrophages are also characterized by the expression of 5-HT2B, an off-target of anesthetics, anti-parkinsonian drugs and Selective Serotonin Reuptake Inhibitors (SSRI) that contributes to 5-HT-mediated pathologies. Since 5-HT2B prevents mononuclear phagocyte degeneration in amyotrophic lateral sclerosis and modulates motility of murine microglial processes, we sought to determine the functional and transcriptional consequences of 5-HT2B activation in human macrophages. Ligation of 5-HT2B by the 5-HT2B-specific agonist BW723C86, which exhibits antidepressant- and anxiolytic-like effects in animal models, significantly modified the cytokine profile and the transcriptional signature in macrophages. Importantly, 5-HT2B agonist-induced transcriptional changes were partly mediated through activation of the Aryl hydrocarbon Receptor (AhR), a ligand-dependent transcription factor that regulates immune responses and the biological responses to xenobiotics. Besides, BW723C86 triggered transcriptional effects that could not be abrogated by 5-HT2B antagonists and impaired monocyte-to-osteoclast differentiation. Therefore, our results demonstrate the existence of a functional 5-HT2B-AhR link in human macrophages and indicate that the commonly used 5-HT2B agonist BW723C86 exhibits 5-HT2B-independent effects. Human peripheral blood monocytes from three independent healthy donors (1, 2 and 3) were isolated by anti-CD14-labeled magnetic microbeads. CD14+ monocytes were cultured for 5 days in RPMI 10% FCS containing M-CSF (M1). After 5 days, cells were maintained in serum-free medium for 48 hours. Then, the resulting macrophages were treated with DMSO (Control), BW723C86 (10mM), SB204741 (1mM) [5-HT2B antagonist] or BW723C86 plus SB204741 during 6 hours.Total RNA from each condition was extracted and purified using the RNA isolation kit (Macheray-Nagel). Labelled RNA was used as hybridization probes on human Agilent SurePrint G3 Human Gene Expression 8x60K (G4851A). All experimental procedures were performed following manufacturer instructions. Microarrays were scanned on an Agilent G2539A scanner. Scanned images and raw data were pre-processed with GeneSpring GX (Agilent).