IRF8 inhibits the activity of C/EBPα to restrain mononuclear phagocyte progenitors from differentiating into neutrophils

During hematopoiesis, myeloid progenitors lose their potential to generate neutrophils when they progress towards monocyte-dendritic cell progenitors (MDPs). However, the mechanism of how this lineage restriction occurs remains poorly understood. Here, we demonstrate that the protein expression of interferon regulatory factor-8 (IRF8), a transcription factor essential for the development of dendritic cells (DCs) and monocytes, sharply increases at the stage of MDPs and remains high in common monocyte progenitors (cMoPs). Irf8–/– MDPs and cMoPs fail to efficiently generate their downstream populations, but instead give rise to neutrophils in vivo, consistent with severe neutrophilia in Irf8–/– mice. Transcriptome analysis predicted that C/EBP(s), the transcription factor(s) essential for the development of neutrophils, is aberrantly activated in Irf8–/– MDPs and cMoPs. In fact, IRF8 physically interacts with C/EBPα, prevents its binding to chromatin, and thereby interferes with its ability to stimulate transcription and neutrophil differentiation. Furthermore, chromatin immunoprecipitation-sequencing analysis revealed that C/EBP DNA binding motifs are significantly enriched in promoter distal-enhancers established by the loss of IRF8, suggesting that the regulation of C/EBPα activity by IRF8 affects the enhancer landscape in MDPs and cMoPs. We also found that a partial inhibition of C/EBP activity in Irf8–/– hematopoietic progenitors remedies the overproduction of neutrophils in vivo, while it does not restore the generation of DCs and monocytes. These results indicate that IRF8 inhibits the transcriptional activity of C/EBP to restrict the neutrophil differentiation potential of mononuclear phagocyte progenitors. During hematopoiesis, myeloid progenitors lose their potential to generate neutrophils when they progress towards monocyte-dendritic cell progenitors (MDPs). However, the mechanism of how this lineage restriction occurs remains poorly understood. Here, we demonstrate that the protein expression of interferon regulatory factor-8 (IRF8), a transcription factor essential for the development of dendritic cells (DCs) and monocytes, sharply increases at the stage of MDPs and remains high in common monocyte progenitors (cMoPs). Irf8–/– MDPs and cMoPs fail to efficiently generate their downstream populations, but instead give rise to neutrophils in vivo, consistent with severe neutrophilia in Irf8–/– mice. Transcriptome analysis predicted that C/EBP(s), the transcription factor(s) essential for the development of neutrophils, is aberrantly activated in Irf8–/– MDPs and cMoPs. In fact, IRF8 physically interacts with C/EBPα, prevents its binding to chromatin, and thereby interferes with its ability to stimulate transcription and neutrophil differentiation. Furthermore, chromatin immunoprecipitation-sequencing analysis revealed that C/EBP DNA binding motifs are significantly enriched in promoter distal-enhancers established by the loss of IRF8, suggesting that the regulation of C/EBPα activity by IRF8 affects the enhancer landscape in MDPs and cMoPs. We also found that a partial inhibition of C/EBP activity in Irf8–/– hematopoietic progenitors remedies the overproduction of neutrophils in vivo, while it does not restore the generation of DCs and monocytes. These results indicate that IRF8 inhibits the transcriptional activity of C/EBP to restrict the neutrophil differentiation potential of mononuclear phagocyte progenitors.