Regulation of an lncRNA irf8 by the Ikzf1/Myb complex drives neutrophil development

As critical executors of the immune system, neutrophils provide an immediate inflammatory response for the clearance of debris and microbes, which is essential for the protection of health. Developmentally, neutrophils and macrophages share common progenitors, whose fate is tightly controlled by transcriptional programs. Dysregulation of these programs causes severe hematological disorders, including neutropenia, neutrophilia, leukemia, and inflammatory disorders. However, the mechanisms underlying the generation of neutrophils through these programs are poorly understood. Here, we revealed that Ikzf1 and Myb were enriched in neutrophils. Overactivation of Ikzf1 promoted neutrophil generation while suppressing macrophage emergence. Conversely, the simultaneous loss of Ikzf1 and Myb, but not the individual mutations, drastically impaired neutrophil production and enlarged the macrophage pool in both zebrafish and mice. Mechanistically, Ikzf1 and Myb formed a complex that targeted irf8 and induced the expression of a long non-coding RNA (lncRNA), irf8-2, through a novel regulatory element. LncRNA irf8-2 biased neutrophil commitment by modulating irf8 dosage via Zfp36l1. The deletion of irf8-2 resulted in defective neutrophil development and enhanced macrophage production. However, a partial ratio of neutrophils and macrophages was restored when Ikzf1, Myb, and Irf8 were all compromised. Overall, our study reveals that Ikzf1 and Myb cooperatively bias neutrophil development against Irf8 via the lncRNA irf8-2 and Zfp36l. This study provides novel insights into the conserved molecular balance between neutrophil and macrophage development during myelopoiesis, with potential implications for understanding and treating myeloid disorders. Tg(coro1a:DsRed) embryos from WT and ikzf1D4+3/D4+3; mybhkz3/hkz3 (DM) embryos at 30 hours post fertilization (hpf) were collected and dissociated using a 1 U mL-1 Papain Dissociation Kit (Worthington) in Dulbeccos phosphate buffered saline (DPBS) at 37 C for 20-30 minutes. Single cell suspensions were generated by gentle trituration, passed through a 40 um cell strainer, and subjected to fluorescence activated cell sorting (FACS). The sorted cells were subsequently processed for Smart seq. Tg(coro1a:DsRed) embryos from WT, Tg(coro1a:HA-ikzf1), Tg(coro1a:FLAG-myb), ikzf1+/+; myb+/+ (WT), and ikzf1D4+3/D4+3; mybhkz3/hkz3 (DM) embryos at 30 hpf were collected and dissociated using a 1 U mL-1 Papain Dissociation Kit (Worthington) in DPBS at 37 C for 20-30 minutes. Single cell suspensions were generated by gentle trituration, passed through a 40 um cell strainer, and subjected to FACS. Approximately 5000 sorted cells were subsequently processed for ATAC seq. Single cell suspensions of FACS sorted coro1a-DsRed cells from more than 30 embryos at 24 hpf were resuspended in DPBS as described in a previous study. The cell suspensions were filtered through a 70 um cell strainer before sorting. The collected coro1a-DsRed positive cells were loaded onto the channels of Single Cell G Chip (v3.1 chemistry, PN1000120).