Macrophages fertilize the soil to promote hematopoietic cell growth

SUMMARY

Inflammatory signals support the birth of hematopoietic stem cells in zebrafish embryos, but their cellular source in mammals is not known. In this issue, Mariani et al. report that macrophages are a primary source of pro-inflammatory signals that promote blood cell formation in mammalian embryos [1].

Hematopoietic stem cells (HSCs) capable of producing the full repertoire of hematopoietic cells for the entire life of the adult animal arise from a specialized subset of endothelial cells, known as hemogenic endothelium, located in the major arteries of the embryo, including in the aorta-gonad-mesonephros (AGM) region [2]. Endothelial cells undergo an endothelial to hematopoietic transition, rounding up and forming intra-aortic cluster cells (IACCs), which contain hematopoietic progenitors (HPs) and stem cells (HSCs). Identifying pathways and signals involved in this process in vivo is necessary to advance efforts to generate HSCs de novo for therapeutic uses.

Several types of HPs emerge from the yolk sac of the embryo before HSCs form in the major arteries [3]. These early progenitors support hematopoiesis in the embryo until the emergence and establishment of HSC-dependent hematopoiesis. Some of these yolk sac progenitors give rise to macrophages that leave the yolk sac to seed many embryonic tissues, eventually giving rise to tissue resident macrophages in the adult [4]. Yolk sac-derived macrophages also play important roles in embryogenesis, for example in clearing apoptotic cells, and for the expansion and enucleation of erythrocytes in the fetal liver [3]. In a study published in this issue of Immunity, Mariana et al. show that yolk sac-derived macrophages also participate in the later formation of HSCs in the AGM region of the mammalian embryo [1].

Recent work in both zebrafish and mouse embryos identified inflammatory signals, including tumor necrosis factor (TNF) and interferons (IFNs), which promote HSC development in the AGM region [5–7]. In zebrafish, loss of neutrophils and macrophages impair HSC formation, but this had not been shown in mammals [5]. Mariani et al. identify macrophages as the most abundant hematopoietic cells in the AGM region of mouse embryos at embryonic day (E) 10.5 [1], when the number of IACCs peaks [8], and suggest they are the primary source of inflammatory signals (Figure 1). In this way, macrophages are the AGM region’s equivalent of earthworms, generating humus in the soil to promote plant growth.

Figure 1: Macrophages stimulate hematopoietic stem and progenitor cell emergence through secreted factors.

Endothelial cells in the AGM region produce the chemokine CX₃CL1, attracting CD206⁺ macrophages. Macrophages in turn promote an endothelial to hematopoietic transition, giving rise to a nascent intra-aortic cluster cell (IACC), through secretion of soluble factors, including TNF and MMPs, and by physically interacting with nascent IACC. Solid black arrows indicate cytokine, chemokine, and MMP production, and the dotted pink line depicts macrophage migration.

The authors show, using several different methods of macrophage depletion, that macrophages promote both HP and HSC formation [1]. Disrupting the CX₃CR1-CX₃CL1 chemokine axis, using CX₃CR1 null mice, prevented macrophage migration to the AGM region and limited HP production. Direct elimination of macrophages by clodronate or with an inhibitor of the colony stimulating factor 1 receptor (CSF1R) reduced both HPs and the number of functional, transplantable HSCs. Taken together, these data clearly demonstrate a requirement for yolk sac-derived macrophages in the early stages of AGM hematopoiesis.

The authors also sought to characterize macrophages in the AGM region capable of promoting hematopoietic development to provide insight into their function. They sub-divided Csf1r-EGFP⁺CD11b⁺F4/80⁺ macrophages into two populations based on CD206 (mannose receptor) expression [1]. Approximately 50% of these macrophages expressed CD206, which they define as AGM-associated macrophages (AGM-aM), because they localized more frequently within the AGM region compared to CD206⁻ macrophages. These data identify CD206 as a marker distinguishing AGM-aMs from other macrophages in the region. This subset of mature CD206⁺ macrophages increased the HP-forming ability of endothelial cells in vitro, whereas phenotypic CD206⁻ macrophages did not. These data suggest that only a subset of macrophages is responsible for promoting hematopoietic cell production from endothelial cells.

The enhanced production of HPs from endothelial cells co-cultured with CD206⁺ macrophages was shown to be contact independent, suggesting the accumulation of soluble factors (humus) is primarily responsible for the increase in HPs during culture. RNA sequencing of the CD206⁺ macrophages revealed a pro-inflammatory signature, consistent with this macrophage subset being the source of inflammatory signals in the developing embryo [1]. Ifna and Ifng, previously identified as promoting HP and HSC development, were not expressed in CD206⁺ macrophages, but Tnf was up-regulated in addition to many other pro-inflammatory signals [5, 6]. TNF could be contributing to the increase in HP formation, but there could be other secreted signals not yet identified that promote hematopoietic development.

CD206 typically marks an anti-inflammatory M2 macrophage in adults. However, in the embryo, CD206⁺ AGM-aMs have a pro-inflammatory signature more reminiscent of a classically activated adult macrophage [1]. Classically activated macrophages in the adult produce pro-inflammatory cytokines with the purpose of eliminating pathogens, while alternatively activated macrophages are associated with anti-inflammatory signals aimed at resolving inflammation and tissue remodeling [9]. The embryonic macrophages described by Mariani et al. seemed to have the transcriptional profile of a pro-inflammatory classically activated macrophage, but they expressed CD206, a classical M2 marker, and were potentially degrading the extracellular matrix to allow for mobilization of HPs and HSCs [1]. Macrophage biology is more complex than classically/alternatively activated designations, and that appears to apply to these CD206⁺ AGM-aMs. The data raise several interesting questions about embryonic macrophages. For example, is macrophage polarization less clear-cut in the embryo compared to the adult? Do embryonic macrophages need to be pro-inflammatory as well as capable of tissue remodeling in order to promote hematopoietic development? Macrophages are polarized in response to external stimuli, and the type of polarization and associated response varies with the stimulus [9]. The macrophages in the embryo had up-regulated inflammatory signatures, indicating they were activated. What stimuli in the sterile environment of the embryo are activating pro-inflammatory signaling in macrophages?

The authors propose that AGM-aMs activate inflammatory programs and stimulate endothelial cells to undergo the transition to hematopoietic cells [1]. However, this hypothesis is based on the pro-inflammatory gene signature of AGM-aMs and not on functional data. Their abundance within the AGM region and localization alongside of emerging hematopoietic cells support the likelihood that macrophages, and perhaps neutrophils as others have shown in zebrafish [5, 6], are initiating cellular sources of pro-inflammatory signals that promote endothelial specification. Specifically interfering with inflammatory signaling components within macrophages and assessing hematopoietic development in vivo would more directly test the authors’ hypothesis.

In addition to producing soluble inflammatory signals, the authors speculated AGM-aMs function through an alternative mechanism by releasing matrix metalloproteinases (MMPs) [1]. In zebrafish, primitive macrophages are essential for HPs and HSCs to migrate from the AGM region to colonize secondary hematopoietic tissues [10]. MMPs secreted by primitive macrophages are necessary to mobilize the HPs and HSCs, as inhibition of MMPs result in accumulation of HPs and HSCs in the AGM region [10]. Mariani et al. discovered that Mmp9 and Mmp13 are both up-regulated in CD206⁺ AGM-aM, providing further support that macrophages have similar functions in supporting HP and HSC emergence and maturation in mouse embryos. Using vital time-lapse microscopy, the authors observed macrophages entering the dorsal aorta and physically interacting with IACCs [1]. They described macrophages intravasating though the aortic wall and “pushing” c-Kit⁺ IACCs toward the lumen. This raises interesting questions about whether physical interaction with macrophages is required in vivo for IACC-maturation and migration to the mammalian fetal liver.

The authors showed that AGM-aM-secretion of soluble factors, such as MMPs and pro-inflammatory cytokines, enhanced HP formation in a CFU assay [1]. However, this assay only detected myeloid progenitor cells with clonogenic capacity, not necessarily multi-potent, self-renewing HSCs. Since the macrophages, themselves, were indeed required for HSC development, it begs the question of what interactions, physical or chemical, are necessary, and when? Do macrophages specify endothelial cells to become hemogenic with HSC potential? Or are they more important after emergence when IACCs have already formed and help HSCs mature? Understanding the mechanism of how exactly macrophages promote HSC formation would be an interesting follow up to this study.

Mariani et al. not only define a functional role for macrophages in mammalian blood development, but assign macrophages as initiators of the hematopoietic cell transition from endothelial cells [1]. The data presented by the authors suggest that macrophages promote HSC formation likely by activation of pro-inflammatory signaling cascades that lead to the release pro-inflammatory cytokines. Endothelial cells receive these cues and undergo hematopoietic specification and HSCs form. Like an earthworm in a garden plot, macrophages turn typically alarming signals into nutrients for endothelial cells to blossom into fruitful HSCs.