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