IRF8 acts in lineage-committed rather than oligopotent progenitors to control neutrophil vs monocyte production

Abstract

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.

Granulocyte-Monocyte Progenitors and Monocyte-Dendritic Cell Progenitors Independently Produce Functionally Distinct Monocytes

“…Consistent with our previous studies (Olsson et al, 2016; Yanez et al, 2015) and the RNA sequencing data, IRF8-GFP was barely detectable in ex vivo GMPs, but strongly expressed by MPs derived from them in M-CSF cultures, whereas e x vivo MDPs and the cMoPs derived from them both strongly expressed IRF8-GFP (Figure 4D). In contrast, Gfi1-tdTomato expression was strongest in the MPs (Figure 4D).…”

“…GMP differentiation then proceeds via a CMP-Flt3 − progenitor, while MDP differentiation does not. GMPs specifically defined as cells with the potential to produce both granulocytes (neutrophils) and monocytes have been convincingly demonstrated in previous studies (especially in colony-forming assays; (Akashi et al, 2000; Yanez et al, 2015)). In contrast, the existence of bipotent or oligopotent MDPs with the ability to yield both monocytes and DCs is more controversial (Sathe et al, 2014), and it is possible that the MDP fraction may comprise a mixture of progenitors committed to either monocyte or DC production.…”

“…“GMPs” were originally defined as LKS − CD34 + FcγR hi cells (Akashi et al, 2000). However, in addition to progenitors with dual granulocyte-monocyte potential (oligopotent or multi-lineage GMPs), this fraction contains granulocyte-committed progenitors (GPs) and monocyte-committed progenitors (MPs or cMoPs) (Akashi et al, 2000; Hettinger et al, 2013; Olsson et al, 2016; Yanez et al, 2015), which we recently reported can be separated on the basis of their expression of Ly6C and CD115 (GMPs are Ly6C − CD115 lo , GPs are Ly6C + CD115 lo , and MPs are Ly6C + CD115 hi (Yanez et al, 2015)). As expected, viSNE analysis confirmed the presence of subsets corresponding to GMPs, GPs and MPs/cMoPs in the LKS − CD34 + FcγR hi fraction of mouse bone marrow (Figure S1A–B).…”

“…(B–C) Ly6C hi , Ly6C − CD43 + and Ly6C − CD43 − subsets were assessed in the bone marrow, blood and spleen of wild-type (B–C), Irf8 -deficient (B) and Nur77 (Nr4a1) -deficient (C) mice with additional gating of CCR2 + cells to identify Ly6C hi monocytes due to the presence of incompletely differentiated Ly6C int monoblasts in Irf8 -deficient mice (Figure S4C and (Yanez et al, 2015)). Data are presented as mean plus standard deviation of 5 mice, and statistical significance was assessed by Student’s t-test (*p<0.05, **p<0.01, ***p<0.001).…”

“…MDPs are widely thought to be derived from GMPs, because both GMPs and MDPs yield monocytes, while MDPs do not give rise to neutrophils (Akashi et al, 2000; Fogg et al, 2006; Hettinger et al, 2013; Yanez et al, 2015). In support of this model, MDP-derived cMoPs (Hettinger et al, 2013) and monocyte-committed progenitors produced by GMPs (named MPs; (Yanez et al, 2015)) appear to be the same cells (on the basis of shared surface marker expression), and adoptively transferred MDPs yield monocytes more rapidly than GMPs (Hettinger et al, 2013; Yanez et al, 2015). However, the relationship between GMPs and MDPs has never been formally evaluated.…”