Key Points• IRF8 does not instruct monocytic lineage specification in oligopotent granulocyte-monocyte progenitors.• IRF8 regulates the survival and differentiation of lineagecommitted progenitors to promote monocyte and suppress neutrophil production.Interferon regulatory factor 8 (IRF8) is a key regulator of myelopoiesis in mice and humans. IRF8-deficient mice exhibit increased neutrophil numbers but defective monocyte and dendritic cell (DC) production. It has therefore been hypothesized that IRF8 regulates granulocyte vs monocyte/DC lineage commitment by oligopotent progenitors. Alternatively, IRF8 could control the differentiation of lineage-committed progenitors. In this study, we defined the role of IRF8 in lineage commitment and neutrophil vs monocyte differentiation using a novel sorting strategy that for the first time allows us to separate oligopotent granulocyte-monocyte progenitors (GMPs) and their lineage-committed progeny: granulocyte progenitors (GPs) and monocyte progenitors (MPs). We show that IRF8 is highly expressed by both GPs and MPs, but not GMPs, and is not required for GP or MP production by GMPs. In fact, IRF8-deficient mice have more GPs and MPs. This is not due to IRF8-mediated suppression of GP and MP production by GMPs, but rather to selective effects in GPs and MPs. We identify roles for IRF8 in regulating progenitor survival and differentiation and preventing leukemic cell accumulation. Thus, IRF8 does not regulate granulocytic vs monocytic fate in GMPs, but instead acts downstream of lineage commitment to selectively control neutrophil and monocyte production.
IntroductionThe hematopoietic system during embryonic development provides two important functions: rapid generation of terminally differentiated blood cells for the survival and growth of the embryo and establishment of a pool of undifferentiated hematopoietic stem cells (HSCs) for postnatal life. To achieve these goals, embryonic hematopoiesis is segregated into multiple waves that occur in several anatomical sites, 1 a process that is broadly conserved in vertebrates. 2,3 The yolk sac is the site of the first wave of embryonic hematopoiesis that generates both primitive red blood cells (RBCs) that deliver oxygen to the embryo and macrophages that assist in tissue remodeling and immune defense. 4 The second wave of hematopoiesis also commences in the yolk sac with the production of a transient pool of erythromyeloid progenitors. Although they lack self-renewal ability and lymphoid potential, they have an important function in fetal hematopoiesis as they rapidly differentiate into mature definitive erythroid and myeloid cells after migration to the fetal liver. 5,6 The third wave of hematopoiesis emerges in the major intra-and extraembryonic arteries, generating HSCs that can both self-renew and differentiate into all blood cell types, including lymphoid cells. HSCs subsequently colonize the fetal liver where they expand before eventually seeding the bone marrow. HSCs emerge in the AGM (aorta-gonad-mesonephros region) and attached vitelline and umbilical arteries, 7-11 the yolk sac, and the placenta. The capacity of the placenta for generation 12 and expansion 13-15 of multipotential hematopoietic stem/progenitor cells has been described recently in both mouse and human, [16][17][18] whereas its potential function as a primitive hematopoietic organ has not been evaluated.The most important products of primitive hematopoiesis that are critical for the survival of the embryo are the primitive RBCs. Experimental evidence suggests that the yolk sac-derived primitive erythroid cells are specified directly from mesoderm with restricted hematopoietic potential, rather than from a multipotential HSC. [19][20][21][22] Primitive red cells differ from definitive red cells not only in their developmental origin, but also in their larger size and distinct globin expression pattern. 23 In mice, primitive RBCs can be identified by expression of ⑀y-globin, 24 which is absent from definitive red cells derived from the fetal liver and the adult bone marrow that express -major globin. 25 In human, primitive red cells uniquely express the ␣-like -globin as well as the -like ⑀-globin. 26 Furthermore, primitive red cells differ from definitive red cells in that they enter circulation as nucleated erythroblasts, whereas the definitive erythroid cells complete maturation and enucleation in their site of origin, the fetal liver 27 or bone marrow, before entering circulation (reviewed in Chasis 28 ). It has been documented that this process occurs in "erythroblast islands" in association with macrophages, which digest the ejected RBC ...
SUMMARYFoxp3+ regulatory T cells (Treg) are essential modulators of immune responses, but the molecular mechanisms underlying their function are not fully understood. Here we show that the transcription factor Blimp-1 is a crucial regulator of the Foxp3+RORγt+ Treg subset. The intrinsic expression of Blimp-1 in these cells is required to prevent production of Th17-associated cytokines. Direct binding of Blimp-1 to the Il17 locus in Treg is associated with inhibitory histone modifications but unaltered binding of RORgt. In the absence of Blimp-1, the Il17 locus is activated, with increased occupancy of the co-activator p300 and abundant binding of the transcriptional regulator IRF4, which is required, along with RORγt, for IL-17 expression in the absence of Blimp-1. We also show that despite their sustained expression of Foxp3, Blimp-1−/− RORγt+IL-17-producing Treg lose suppressor function and can promote intestinal inflammation, indicating that repression of Th17-associated cytokines by Blimp-1 is a crucial requirement for RORγt+ Treg function.
Summary Several groups have shown that detection of microbial components by Toll-like receptors (TLRs) on hematopoietic stem and progenitor cells (HSPCs) instructs myeloid cell generation, raising interest in the possibility of targeting TLRs on HSPCs to boost myelopoiesis. However, although “TLR-derived” cells exhibit myeloid cell characteristics (phagocytosis, cytokine production, antigen presentation), it isn’t clear whether they are functionally equivalent to macrophages derived in the absence of TLR activation. Our in vitro and in vivo studies show that macrophages derived from mouse and human HSPC subsets (including stem cells) exposed to a TLR2 agonist prior to or during macrophage differentiation produce lower levels of inflammatory cytokines (TNF-α, IL-6 and IL-1β) and reactive oxygen species (ROS). This is in contrast to prior exposure of differentiated macrophages to the TLR2 agonist (“tolerance”), which suppresses inflammatory cytokine production, but elevates ROS. Soluble factors produced following exposure of HSPCs to a TLR2 agonist can also act in a paracrine manner to influence the function of macrophages derived from unexposed HSPCs. Our data demonstrate that macrophage function can be influenced by TLR signaling in the HSPCs from which they are derived, and that this may impact the clinical utility of targeting TLRs on HSPCs to boost myelopoiesis.
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