Irf8 regulates macrophage versus neutrophil fate during zebrafish primitive myelopoiesis

Abstract

IntroductionMyeloid cells or phagocytes are a subtype of leukocytes that play essential roles in the host defense, embryogenesis, organogenesis, and tissue regeneration. [1][2][3] In vertebrates, myeloid cells are classified into 2 major lineages: polymorphonuclear and mononuclear lineages, which acquire different morphologies during development and exert overlapping but distinctive biologic functions. Polymorphonuclear phagocytes consist of neutrophils, eosinophils, basophils, and mast cells. They are the key effectors of inflammatory response on pathogen infection and tissue injury. On the other hand, mononuclear phagocytes, which include circulating macrophages, dendritic cells, and tissue-resident macrophages (osteoclasts in the bone, microglia in the brain, and Kupffer cells in the liver), not only play important roles in inflammatory response but also participate in organogenesis and tissue regeneration. 2,3 Despite differences in morphology and biologic function, polymorphonuclear and mononuclear phagocytes are thought to derive from a common myeloid-restricted population termed neutrophil-macrophage progenitors. 4 These multipotent neutrophil-macrophage progenitors then differentiate to macrophage-dendritic cell progenitors, neutrophil-macrophage progenitors, and basophil-mast cell bipotent progenitors, which in turn undergo terminal differentiation to produce mature dentritic cells, macrophages, neutrophils, basophils, and mast cells. 5 This developmental process is tightly controlled, and dysregulation of phagocyte development and function is associated with several human diseases, including cancer, autoimmune disorders, and neurodegenerative disorders.Interferon regulatory factor-8 (IRF8), also known as the interferon consensus sequence-binding protein, was first identified through screening mouse expression libraries with interferon consensus sequence as a probe. 6 It encodes a transcription factor of IRF family, which contains a highly conserved N-terminal DNA-binding domain and a less conserved C-terminal IRF association domain. 7 Among the 9 members of the mammalian IRF family, IRF4 and IRF8 share the highest similarity in protein sequence and they are predominantly expressed in lymphocytes, macrophages, and dendritic cells. 8 The importance of IRF8 in hematopoiesis is first revealed by genetic studies in IRF8 knockout (IRF8-null) 9 and BXH-2 mutant mice, which carry a loss-of-function mutation in the IRF association domain of IRF8 protein. 10 Both IRF8-null and BXH-2 mutant mice have chronic myeloid leukemia with a profound increase of neutrophil number. 9,10 In addition, loss-of-function mutation in IRF8 gene in these animals also causes a severe reduction of macrophages and dendritic cells. [10][11][12][13] These in vivo studies reveal that IRF8 is essential for myeloid progenitor differentiation toward macrophages but is dispensable for neutrophil development during adult murine myelopoiesis. Subsequent study shows that forced expression of IRF8 in IRF8-deficient myeloid progenitor cell line p...

Live Imaging Reveals Differing Roles of Macrophages and Neutrophils during Zebrafish Tail Fin Regeneration

“…The consequential colonization of the wound by the local resident macrophages/neutrophils, distant/circulation-recruited neutrophils, and distant/circulation-derived macrophages is partially in agreement with the previous study in mice, which suggested that the local resident macrophages may facilitate the activation and recruitment of the distant/circulation-derived neutrophils and macrophages (44). However, contrary to this notion, our study showed that the activation and recruitment of both local resident and distant/circulation neutrophils are not affected in the irf8 morphants, in which resident macrophages and circulating monocytes are absent (28), indicating that the local resident macrophages are not essential for the activation and recruitment of distant/circulation-derived neutrophils. Further study is needed to clarify the role of the local resident macrophages.…”

“…To examine the co-staining of GFP and DsRed, embryos were first stained with goat anti-GFP and rabbit anti-DsRed antibody (1:250, 4°C, overnight) and subsequently visualized by Alexa Fluor 488 donkey anti-goat (1:400, 4°C, overnight) and Alexa Fluor 555 donkey anti-rabbit (1:400, 4°C, overnight). Similar methods were used for co-staining of GFP and Lcp protein and of GFP and phosphohistone 3 (pH3) (28). Anti-Lcp and anti-pH3 (Upstate) antibody was visualized by Alexa Fluor donkey anti-rabbit 555 (1:400, 4°C, overnight) (Molecular Probes).…”

“…To support this hypothesis, we depleted macrophages by knocking down irf8 (interferon regulatory factor 8) in zebrafish by specific MOs (28). In the irf8 MO-injected embryos (morphants), neutrophils responded normally to tail amputation (supplemental Fig.…”

“…Injection-0.6 mM irf8 MOsp (5Ј-AATGTTTCGCT-TACTTTGAAAATGG-3Ј) or 0.3 mM irf8 MOatg (5Ј-TCAGTCTGCGACCGCCCGAGTTCAT-3Ј) and control morpholino-oligonucleotide were injected into the one-cell stage embryos (28).…”

miR-142-3p regulates the formation and differentiation of hematopoietic stem cells in vertebrates

“…Inflammatory activation of early HSCs has been shown to be evolutionarily conserved [38], and a recent report by Hall et al [31] demonstrated that, in zebrafish, Gcsfr-NO signaling is required for the expansion of HSCs following infection. Among the irf family members, irf8 was shown to be required for myeloid differentiation into macrophages as opposed to neutrophils [39] and for the regulation of B-cell lineage specification and differentiation [40]. Here, we reveal that knocking down miR-142a-3p expression in zebrafish upregulated the expression of an irf family member, irf7, which might be responsible for the decreased HSC population in the miR-142a-3p morphants.…”