Monocytes differentiate into macrophages or dendritic cells along two alternative paths controlled by distinct regulatory networks

During inflammation, monocytes differentiate within tissues into macrophages (mo-Mac) or dendritic cells (mo-DC). Whether these two progenies derive from alternative differentiation pathways or represent different stages along a continuum remains unclear. Here we addressed this question using temporal single-cell RNA sequencing in an in vitro model allowing the simultaneous differentiation of human mo-Mac and mo-DC. We evidenced divergent differentiation paths, with a fate decision occurring within the first 24 hours. We confirmed this result in vivo using a mouse model of peritonitis. Using a computational approach, we identified candidate transcription factors potentially involved in monocyte fate commitment. We demonstrated that IRF1 is necessary for mo-Mac differentiation, independently of its transcriptional control of interferon-stimulated genes. In addition, we validated the transcription factors ZNF366 and MAFF as regulators of mo-DC development. Our results indicate that mo-Mac and mo-DC represent two alternative cell fates requiring distinct transcription factors for their differentiation. Overall design: Kinetic of monocyte differentiation by scRNAseq. We performed scRNAseq of monocytes cultured with a cytokine cocktail that yields to the differentiation of monocytes-derived macrophages and dendritic cells. We combined this technology to HASH-sequencing in order to measure these data simultaneously in two healthy human donors and at different time points.