Author response: Fetal liver macrophages contribute to the hematopoietic stem cell niche by controlling granulopoiesis

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Full text Figures and data Peer review Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Article and author information Metrics Abstract During embryogenesis, the fetal liver becomes the main hematopoietic organ, where stem and progenitor cells as well as immature and mature immune cells form an intricate cellular network. Hematopoietic stem cells (HSCs) reside in a specialized niche, which is essential for their proliferation and differentiation. However, the cellular and molecular determinants contributing to this fetal HSC niche remain largely unknown. Macrophages are the first differentiated hematopoietic cells found in the developing liver, where they are important for fetal erythropoiesis by promoting erythrocyte maturation and phagocytosing expelled nuclei. Yet, whether macrophages play a role in fetal hematopoiesis beyond serving as a niche for maturing erythroblasts remains elusive. Here, we investigate the heterogeneity of macrophage populations in the murine fetal liver to define their specific roles during hematopoiesis. Using a single-cell omics approach combined with spatial proteomics and genetic fate-mapping models, we found that fetal liver macrophages cluster into distinct yolk sac-derived subpopulations and that long-term HSCs are interacting preferentially with one of the macrophage subpopulations. Fetal livers lacking macrophages show a delay in erythropoiesis and have an increased number of granulocytes, which can be attributed to transcriptional reprogramming and altered differentiation potential of long-term HSCs. Together, our data provide a detailed map of fetal liver macrophage subpopulations and implicate macrophages as part of the fetal HSC niche. Editor's evaluation Using single-cell sequencing, high-resolution imaging, and inducible genetic deletion of yolk-sac (YS) derived macrophages, the authors present a useful map of fetal liver macrophage subpopulations and provide important data demonstrating that heterogeneous fetal liver macrophages regulate erythrocyte enucleation, interact physically with fetal HSCs, and may regulate neutrophil accumulation in the fetal liver. These important findings provide a solid foundation for further investigating the effects of macrophages on HSC function during fetal hematopoiesis and into adulthood and will be useful for the field of macrophage biology and developmental hematopoiesis. https://doi.org/10.7554/eLife.86493.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Macrophages are found in all adult organs, where they perform essential functions during inflammatory responses as well as in tissue homeostasis, such as tissue remodeling, phagocytosis of apoptotic cells, and production of cytokines and growth factors. Work in mice showed that most macrophages originate from erythro-myeloid progenitors (EMPs) in the yolk sac and are long lived, and that their maintenance in many adult tissues does not rely on definitive hematopoiesis (Gomez Perdiguero et al., 2015; Hoeffel et al., 2015; Mass, 2018). All developing tissues are initially colonized by circulating pre-macrophages (pMacs), which immediately differentiate into tissue-specific macrophages (Mass et al., 2016; Stremmel et al., 2018), therefore, macrophages are an integral part of organogenesis. In adult tissues, macrophages inhabit distinct anatomical niches within organs, for example, the lung harbors alveolar and interstitial macrophage populations (Aegerter et al., 2022), while macrophages in the adult liver are divided into Kupffer cells, liver capsular, central vein, and lipid-associated macrophages (Guilliams and Scott, 2022). Within these niches, resident macrophages adapt to their tissue environment and perform specific tasks to maintain organ function, such as mucus clearance by alveolar macrophages or phagocytosis of red blood cells by Kupffer cells (Mass et al., 2023). However, the role of macrophages in organ development and function as well as their heterogeneity during embryogenesis is less well understood. One of the few well-known functions of fetal macrophages is their involvement in erythroblast maturation. In the fetal liver, erythroblastic island (EI) macrophages serve as niches for erythroblasts. During erythropoiesis, primitive and definitive erythroblasts directly interact with EI macrophages, where they undergo final steps of maturation, including expelling of their nucleus, which is phagocytosed by EI macrophages (Palis, 2016; Palis, 2014). Previous studies identified EI macrophage heterogeneity in the fetal liver at embryonic day (E)13.5/E14.5 (Li et al., 2019; Mukherjee et al., 2021; Seu et al., 2017). EI macrophages were shown to express different levels of cell adhesion proteins such as Vcam1, CD169, and CD163, as well as other proteins that are important for their function as EI niche (e.g., Epor, Klf1, EMP, and DNaseII), thereby promoting erythropoiesis (Li et al., 2019; Mariani et al., 2019; May and Forrester, 2020; Mukherjee et al., 2021). In addition to erythropoiesis, the E13.5/E14.5 fetal liver is at the peak of hematopoiesis, providing a niche for the expansion and differentiation of other hematopoietic stem and progenitor cells (HSPCs) (Lewis et al., 2021). Thus, the liver is a complex cellular interaction network where the role of macrophages in other hematopoietic developmental processes, such as myelopoiesis, is not fully understood. Recent evidence suggests that one of the core macrophage functions is the support of stem and progenitor cell functionality in different tissues. For instance, pericryptal macrophages in the gut interact with epithelial progenitors, thereby promoting their proliferation and differentiation via Wnt, gp130, TLR4, or NOX1 signaling (Delfini et al., 2022). In the bone marrow, distinct macrophage subpopulations contribute directly and indirectly to the hematopoietic stem cell (HSC) niche. Osteoclasts are a highly specialized EMP-derived macrophage population responsible for bone resorption, whose function is necessary to generate the bone marrow and, thereby, the postnatal HSC niche (Jacome-Galarza et al., 2019). Co-culture experiments using different combinations of hematopoietic cells indicate that osteomacs support the HSC niche in synergy with megakaryocytes (Mohamad et al., 2017). DARC+ macrophages are in direct contact with CD82+ long-term (LT)-HSCs in the endosteal and arteriolar niches, where they contribute to LT-HSC dormancy via maintenance of CD82 expression (Hur et al., 2016). Additional macrophage depletion studies via clodronate and diphtheria toxin indicate that CD169+ macrophages in the bone marrow promote HSC retention by acting specifically on the Nestin+ HSC niche (Chow et al., 2011), as well as steady-state and stress erythropoiesis (Chow et al., 2013). During embryogenesis, macrophages have been shown to be important for the development and proliferation of hematopoietic stem- and progenitor cells in mice. In the mouse, the first HSCs develop from the aorta–gonad–mesonephros (AGM) region starting at E10.5. Here, a CD206+ macrophage population actively interacts with nascent and emerging intra-aortic HSCs (Mariani et al., 2019). Macrophage depletion studies via clodronate liposomes and the Csf1r inhibitor BLZ945 using AGM explants indicate that the presence of macrophages in the AGM is influencing HSC production (Mariani et al., 2019). Furthermore, co-culture studies of a mixture of hematopoietic stem- and multipotent-progenitor cells (HSC/MPP) with macrophages isolated from the fetal liver as well as clodronate depletion studies during embryogenesis suggest that fetal liver macrophages promote HSC/MPP proliferation (Gao et al., 2022). Indeed, immunofluorescent stainings of F4/80+ macrophages and CD150+ cells (Gao et al., 2022) indicate that macrophages could also be part of the LT-HSC niche in the fetal liver, thereby influencing the proliferation and differentiation of stem cells at the top of the HSC hierarchy. Studying stem cell niches and defining the molecular factors that promote stem cell proliferation and differentiation are essential to build a robust and reproducible in vitro system that may serve as a universal source of functional stem cells and/or immune cells for therapeutic purposes. This has already been achieved for induced pluripotent stem cells (iPSCs), which can be infinitely expanded and differentiated into the cell type of interest, thereby representing a safe product to treat diseases (Yamanaka, 2020). In contrast, a universal and well-defined culturing protocol for LT-HSCs allowing for continuous expansion is lacking (Kumar and Geiger, 2017; Wilkinson et al., 2020). Production of LT-HSCs from iPSCs represents a promising alternative, however, differentiation protocols without ectopic transcription factor expression have not been established yet (Demirci et al., 2020). This may be due to the complex developmental programming of adult LT-HSCs that have experienced distinct niche signals during their migration from AGM via the fetal liver to the bone marrow, resulting in functional differences observed in fetal versus mature HSCs (Arora et al., 2014). Growing evidence, mainly provided by in vitro and ex vivo studies in combination with macrophage depletion via clodronate (Gao et al., 2022; Mariani et al., 2019), suggests that macrophages play an essential role in HSC development and maintenance during embryogenesis. However, it remains to be investigated whether specific macrophage populations in the fetal liver contribute to LT-HSC stem-cell ness or differentiation via factors they produce in vivo. Given the observations implicating macrophages in HSC functionality, we characterized the heterogeneity and ontogeny of fetal liver macrophage populations, providing a comprehensive macrophage atlas of the fetal liver at E14.5. Furthermore, using a conditional mouse model to deplete macrophages in vivo, we established fetal liver macrophages as important modulators of LT-HSC differentiation capacity into granulocytes. Results The fetal liver harbors heterogeneous macrophage populations To investigate fetal liver macrophage heterogeneity, we performed single-cell RNA-sequencing on sorted CD11blow/+ cells at E14.5 (Figure 1—figure supplement 1A). To isolate macrophages and macrophage progenitors for further downstream analyses from this myeloid population, we clustered all cells and overlayed a pMac signature (Mass et al., 2016). Out of eleven clusters, four clusters were chosen (Figure 1—figure supplement 1B, C , see Methods) and analyzed further. To further select specifically macrophages, the same procedure was performed twice on the re-clustered cells using a signature enriched in fetal macrophages when compared with EMPs (Figure 1—figure supplement 1D), resulting in 18 clusters (Figure 1—figure supplement 1E). Cells that either expressed macrophage precursor genes (Clec7a, Ccr2, Cx3cr1, Csf1r), pan-macrophage genes (Mrc1, Adrgre1, Siglec1, Msr1, Cd63), and/or liver macrophage-specific genes (Timd4, Clec4f, Vcam1) were enriched in clusters 1, 2, 7, 8, 9, and 11, which were chosen for further downstream analysis (Figure 1A, B, Figure 1—figure supplement 1F). The predominant expression of Ly6c2, Ly6g, Cxcr2, and Cd33 in the remaining clusters (Figure 1—figure supplement 1F) indicated their monocyte/granulocyte or myelomonocytic precursor cell identity, respectively, and were therefore excluded from further analysis. Figure 1 with 2 supplements see all Download asset Open asset Characterization of fetal liver macrophage populations. (A) Single-cell RNA-sequencing (scRNA-seq) analysis of wildtype CD11blow/+ cells isolated from a fetal liver at developmental day (E)14.5. Clusters of possible macrophage subsets identified by a monocyte/macrophage signature (see Figure 1—figure supplement 1) are visualized through Uniform Manifold Approximation and Projection (UMAP). (B) Expression of selected macrophage- and macrophage progenitor-specific genes in clusters from (A). (C) Violin plots of highly expressed genes within the clusters from (A). (D) Developmental trajectory analysis using Partition-based graph abstraction (PAGA) method (left) and pseudotime analysis (right) of the identified clusters in (A), excluding cluster 9. (E) Flow-cytometry analysis of CD11blow/+ cells with a macrophage signature, isolated from a fetal liver at developmental day E14.5. Cell surface marker expression was used to generate unbiased clusters using UMAP. (F) Heatmap of relative protein expression and cell size parameters used in the flow-cytometry analysis in (E). (G) Flow-cytometry analysis of E14.5 fetal liver macrophages using three different fate-mapping mouse models. CD11blow/+ cells with a macrophage signature were analyzed as shown in (E, F), resulting in similar cluster distribution (see Figure 1—figure supplement 2). YFP+ cells from the Tnfrsf11aCre; Rosa26YFP model (green) indicate a yolk sac origin. Ms4a3Cre; Rosa26YFP (pink) and Cxcr4CreERT; Rosa26YFP with 4-hydroxytamoxifen (4-OHT) injection at E10.5 (gray) indicate a monocytic and hematopoietic stem cell origin, respectively. Circles represent individual mice. (H) Simplified gating strategy to identify macrophage clusters in E14.5 livers using flow cytometry. Figure 1—source data 1 Quantification of fate-mapped macrophages populations for Figure 1. https://cdn.elifesciences.org/articles/86493/elife-86493-fig1-data1-v2.xlsx Download elife-86493-fig1-data1-v2.xlsx Cluster 9 expressed almost exclusively erythroblast-specific genes (Hba-a2, Sptb, Trim10, Nxpe2, Snca, Epb42) (Figure 1B,C) and was identified as EI macrophages, which have been recently described as doublets of erythroblasts and macrophages (Popescu et al., 2019) or erythroblasts with cell remnants on their surface since macrophages frequently adhere to other cells (Millard et al., 2021). Clusters 7 and 8 showed the highest expression of bona fide macrophage genes such as Csf1r, Mrc1, and Timd4 but were distinct in their expression of other macrophage markers (e.g., Marco, Lyve1) (Figure 1B, C, Figure 1—figure supplement 1E). Cells in cluster 2 expressed the highest levels of Ccr2, Clec7a, Clec4a, Il1b, and S100a6 (Figure 1B, C) besides some of the macrophage-specific genes, hinting toward an inflammatory state of this putative macrophage population. Clusters 1 and 11 showed a low expression of core macrophage genes compared to clusters 2, 7, and 8, suggesting a precursor stage (Figure 1B). However, cluster 11 was distinct from cluster 1 with a high expression of granule-related genes, such as Mpo, Elane, and Ctsg (Figure 1C), which may indicate a granulocytic rather than a macrophage precursor state. To test these hypotheses and predict developmental trajectories, we performed a Partition-based graph abstraction (PAGA) analysis (Wolf et al., 2019) after excluding cluster 9 due to their doublet identity. Here, cluster 1 expression represents a progenitor state, thus, is the center of the network, which was confirmed by pseudotime analysis (Figure 1D). The other clusters fall into distinct nodes, with clusters 7 and 8 showing substantial similarity as indicated by the edge thickness (Figure 1D). In summary, our scRNA-seq analysis indicates the presence of at least three macrophage states (clusters 2, 7, and 8), which have distinct phenotypes. To validate macrophage heterogeneity on the protein level, we performed a high-dimensional flow-cytometry analysis on CD11blow/+ cells (Figure 1—figure supplement 2A). Similar to the scRNA-seq enrichment, we visualized all myeloid cells using UMAP, clustered them, and overlayed a macrophage signature (F4/80, Tim4, Cx3cr1, Vcam1, CD169, CD206, Figure 1—figure supplement 2B). This resulted in seven clusters, which we analyzed further (Figure 1E). Hierarchical clustering of all clusters expressing macrophage and macrophage precursor proteins confirmed the presence of three F4/80high macrophage populations (clusters A–C, Figure 1F). As already observed on the transcriptional level, cluster D likely represents Ter119+ erythroblasts with macrophage cell remnants, as indicated by the increased cell size and granularity determined via forward scatter (FSC) and side scatter (SSC), respectively (Figure 1F). To correlate precursor and macrophage clusters identified by transcriptional and protein analyses, a correlation matrix between the scRNA-seq and flow-cytometry datasets was calculated based on gene expression corresponding to the presence of the respective antigen used in our flow-cytometry panel (Figure 1—figure supplement 2C). Here, scRNA-seq precursor clusters 1 and 11 corresponded highly to clusters F and G, which expressed high levels of CD45, CD115, and Clec4f but were low in F4/80 and Tim4 expression. In contrast, scRNA-seq clusters 2 and 7 represented cluster A while cluster 8 correlated mostly with clusters B and C, supporting the notion of three distinct macrophage populations in the fetal liver at E14.5, in addition to EI macrophages. Fetal liver macrophages originate from yolk sac progenitors Next, we addressed the ontogeny of the macrophage clusters using Rosa26LSL-YFP mice crossed to Tnfrsf11aCre for detection of pMac-derived cells (Mass et al., 2016), to Ms4a3Cre for detection of monocyte-derived cells (Liu et al., 2019), and to the inducible Cxcr4CreERT with 4-hydroxytamoxifen injection at E10.5 labeling all cells of the definitive hematopoiesis wave (Werner et al., 2020). We confirmed the presence of all three macrophage clusters and the Ter119+ EI macrophage cluster in all mouse models (Figure 1—figure supplement 2D). HSC-derived definitive erythroblasts in cluster D were efficiently fate-mapped using the Cxcr4CreERT model, validating that these were cell doublets or erythroblasts with cell remnants on their surface since they also showed low labeling of the other fate-mapping models that label macrophages/monocytes (Figure 1G). The remaining clusters were YFP+ only in the Tnfrsf11aCre model, demonstrating that all fetal liver macrophages at E14.5 derive from pMacs. Using the distinct expression of Vcam1, CD206, and CD169 in clusters A–C and their difference in cell size allowed us to develop a simple gating strategy to distinguish these macrophage populations (Figure 1H). In summary, using a hypothesis-driven analysis of CD11blow/+ cells, we define, in addition to the already well-known EI macrophage population, distinct macrophage populations in the fetal liver that are yolk sac derived. Fetal liver macrophage subpopulations display distinct transcriptional programs Next, we set out to investigate whether the macrophage heterogeneity we defined at E14.5 could indicate additional macrophage states besides serving as EI macrophages, therefore, resulting in other functions than phagocytosing erythroblast nuclei. To this end, we first performed a gene ontology (GO) term analysis on the top expressed 100 genes, comparing each cluster from the scRNA-seq analysis to all other clusters. Here, cluster 2-specific genes fell into the terms 'cell chemotaxis', 'positive regulation of inflammatory response', and 'cytokine-mediated signaling pathway' (Figure 2A), indicating the activated inflammatory state already observed in the top 5 expressed genes (Figure 1C). In contrast, cluster 7-related genes were significant for the GO terms 'positive regulation of lipid localization', 'ameboidal-type cell migration', and 'apoptotic cell clearance'. Also cluster 8 expressed genes belonging to the GO term 'positive regulation of lipid localization' that partially overlapped with cluster 7 (e.g., Apoe, Lpl) (Supplementary file 1). Additional cluster 8-specific terms were 'macrophage activation' and 'cell junction disassembly' (Figure 2A). This analysis indicated that, albeit somewhat similar, the distinct macrophage populations might exert distinct functions in the fetal liver. This was confirmed by the cluster-specific expression of selected ligands indicating that the distinct cellular states may result in distinct paracrine signaling activity (Figure 2B). Intersecting the CellTalk database information (Shao et al., 2021) with the complete set of genes expressed by the three macrophage states revealed 208 potential macrophage-derived secreted ligands (Supplementary file 2). Figure 2 Download asset Open asset Transcriptional program and paracrine signaling of fetal liver macrophages. (A) Gene set enrichment analysis (GSEA) of final macrophage clusters 2, 7, and 8 was performed on the differentially expressed genes of each cluster. (B) Expression of selected ligands in the final macrophage clusters. (C) Interaction and functional network of expressed ligands on the identified macrophage clusters 2, 7, and 8. Each hub with a color indicates the function of the ligands, while the edges show the interaction between them. (D) Potential ligand–receptor interactions between macrophages and long-term hematopoietic stem cells (LT-HSCs). The gene names on the bottom of the plot are expressed ligands in macrophage clusters 2, 7, and 8. Gene names on the top are expressed receptors on LT-HSCs at E14.5. Each ligand can target several receptors which are indicated with the same color. We next used the 208 ligand candidates and performed a gene set enrichment analysis, and visualized the gene/GO term relationships in a network (Figure 2C). This analysis pointed to additional functionality of the three macrophage states beyond erythropoiesis, which included regulation of hemopoiesis and stem cell development together with chemotaxis and vasculature development, mechanisms that could shape the stem cell niche. Macrophages contribute to the stem cell niche, particularly in the bone marrow, under inflammatory conditions (Seyfried et al., 2020). Yet, evidence for macrophage-derived molecules involved in the direct cell crosstalk controlling stem cell maintenance and differentiation in the mouse fetal liver is missing. Therefore, we sought to explore potential signaling events between macrophages and HSCs to determine whether macrophage-derived factors might modify HSC function during steady state. Thus, we sequenced LT-HSC at E14.5 and, leveraging our scRNA-seq macrophage dataset (see Methods), uncovered potential ligand–receptor interactions between macrophage-derived ligands and LT-HSCs in the fetal liver (Figure 2D). Many of the ligands are well-known players in the stem cell niche, for example, Kitl, Igf1, Tnf, Tgfb1, and Fn1, which have been reported to directly or indirectly promote the expansion of et al., et al., 2022; et al., 2013). In summary, our ligand–receptor interaction analyses suggest that macrophages express HSC niche factors and, thereby, may actively contribute to LT-HSC functionality in the fetal liver. Macrophages interact directly with LT-HSCs We next whether the potential paracrine signaling between macrophages and LT-HSCs in the fetal liver via direct To test we first performed analyses on E14.5 liver by macrophages with and F4/80 and LT-HSCs with indicating a direct interaction between these cells (Figure Next, we performed stainings on livers at E14.5 using different stem and erythroblast-specific We found EI macrophages that were by Ter119+ as but frequently observed interactions of macrophages with progenitors and CD150+ LT-HSCs (Figure analysis of macrophage in combination with CD150+ and cell (Figure showed that CD150+ cells were to macrophage as indicated by an increased within a of compared to cells (Figure Quantification of the to an cell that CD150+ cells to macrophages compared to cells, suggesting a of CD150+ cells macrophages (Figure Figure Download asset Open asset Macrophage interaction with CD150+ hematopoietic stem cells (A) of E14.5 fetal liver with and represents (B) of E14.5 fetal liver with Tim4, and The first the interaction between EI macrophages by Ter119+ erythroblasts. The and the interaction between macrophages and progenitor cells, including CD150+ long-term HSCs (LT-HSCs). The on the indicate the and which are shown on the and bottom represents 5 (C) of a E14.5 fetal liver with and as indicating represents (left) or of CD150+ and cells to the next macrophage (D) and of cells to macrophages for CD150+ and for (E). Figure data 1 between CD150+ or cells and cells for Figure Download To determine whether LT-HSCs interact with distinct macrophage populations, we cellular interactions via by high-dimensional et al., 2021; et al., 2022; et al., Figure CD45, Cx3cr1, and Tim4 were used as pan-macrophage allowing the of cluster macrophages, cluster CD206+ macrophages, cluster CD169+ macrophages, and the most cluster Ter119+ EI macrophages (Figure Figure supplement 1A). we the distribution of the four macrophage clusters in the different liver using a showing that all macrophage clusters are found the tissue (Figure CD150+ HSC were also the fetal liver but showed a the liver (Figure Similar were via cellular of the using a with a of and a map where CD150+ HSCs, represented by were mainly found the liver of the and the liver (Figure supplement 1B). Next, we used a approach to spatial interactions between macrophage clusters and HSCs within a of LT-HSCs showed the highest correlation with macrophages from cluster C representing CD169+ macrophages (Figure and the correlation with cluster D of cells CD150+ HSC in a also revealed that LT-HSCs were preferentially by CD169+ macrophages C, Figure with identified cells belonging to the other macrophage populations (Figure Figure with 2 supplements see all Download asset Open asset Macrophage heterogeneity and their interaction with hematopoietic stem cells A of a fetal liver from an E14.5 wildtype was with a of the field of is shown in (A) and showing of the cells from clusters are shown in in the indicate macrophages from the corresponding cluster. represent in the and in the (C) from (B) after cell using the HSCs, blood and cells from the four macrophage clusters as (D) analysis of interactions between cells from macrophage clusters, HSCs, and blood in the fetal liver within a of represent the calculated (E) of the interaction of HSCs with the macrophage clusters. indicate the and indicate the macrophages from the corresponding cluster. is shown in color. represents (F) number of cells from the four macrophage clusters within a of from one selected Each represents one in the plot represent the analysis of Figure data 1 of CD150+ cells within each macrophage in a Download As erythroblasts are the most cell we whether clusters A–C at least as EI macrophages. To this end, macrophages of each cluster were and the direct interaction with Ter119+ cells was cells belonging to cluster D EI showed as cluster A macrophages, cluster B macrophages, and cluster C macrophages, not interact with Ter119+ erythroblasts (Figure supplement The CD206+ cluster A was the with the least