Macrophage subsets provide distinct niches for normal and clonal hematopoiesis Free

Hematopoietic stem cells (HSCs) are tightly regulated by the bone marrow (BM) niche to ensure blood homeostasis. Dysfunction of the niche contributes to disrupted proliferation, differentiation and self-renewal of HSCs in clonal hematopoiesis. Macrophages are known to support erythropoiesis and promote HSC retention. We recently showed that macrophages also regulate HSC fate decisions by controlling local iron availability, and confer residency through a membrane transfer process termed trogocytosis (Zhang D, Cell Stem Cell 2022; Gao X, Science 2024). However, whether these functions of BM macrophages are mediated by the same or distinct sub-populations remain largely unclear.BM macrophages have no clear separation of subsets, which has been an intriguing puzzle for years. The trogocytosis phenomenon suggest that “macrophages” identified by common markers, such as F4/80, contain cells that acquire those markers via transfer (Gao X, Science 2024). To solve this problem, we broadly surveyed the expression of surface markers, and found that the combination of CD169 and MerTK separated F4/80⁺ cells into three distinct sub-populations, R1, R2 and R3. Notably, macrophage-specific genes, such as CD163, Hmox1, and MerTK, were exclusively expressed in R1 cells. To assess the functional roles, we challenged mice with 5-fluorouracil (5FU) to trigger RBC clearance in the BM (Zhang D, Cell Stem Cell 2022), and observed phagocytic events only in R1, suggesting that R1 cells are bona fide macrophages in the BM. We further performed single-cell RNA-seq, and resolved R1 cells into 6 transcriptionally distinct clusters with divergent functional specialization. Flow cytometry analyses validated expression of selected markers for these clusters, such as CD163 and MHC-II, yielding gating strategies for R1 subsets.The heterogeneity of BM macrophages may arise from distinct embryonic or hematopoietic origins. To clarify this, we first analyzed the developmental kinetics of R1 cells, and observed that they readily emerged at the embryonic stage. By transplantation assays, we found that total BM and HSCs were able to replace ~80% of R1 cells. Parabiosis experiments further revealed that R1 cells could be replenished via the circulation. These results suggest that definitive hematopoiesis contributes to the development and turnover of BM macrophages. To identify which progenitors, such as granulocyte-monocyte progenitors (GMP), Ly6C⁺and Ly6C⁻ monocytes, contribute to the development of R1 subsets, we traced reporter expression using Ms4a3-Cre, CCR2- and Cx3CR1-CreER; tdTomato mice (Liu Z, Cell 2019). We found that the Ms4a3 model labeled ~50% of both CD163⁺ and CD163^- subsets, suggesting mixed origins from GMPs and other progenitors. Further, the CCR2 and CX3CR1 models labeled the CD163^- and CD163⁺ subsets respectively, suggesting that Ly6C⁺and Ly6C⁻ monocytes contributed differentially to macrophage subsets in the BM.Distinct macrophage subsets may provide niches for the interactions with different cell types. To test this hypothesis, we developed whole-mount immunofluorescence panels in sternum, to visualize the anatomical associations between R1 subsets and HSPCs. We first analyzed the anatomy of erythroid progenitors, and observed synergistic interactions between sinusoids and the CD163⁺ subset in regulating stress erythropoiesis. Next, we observed that HSCs were anatomically associated with the CD163⁺ subset. Upon 5FU challenge, HSCs migrated away from the CD163⁺ subset at later stages of regeneration, which strongly correlated with proliferative activation. We further expanded our analyses to clonal diseases using PU.1-URE^-/- mice, which exhibited ~80% downregulation of PU.1, leading to the development of myeloid malignancies. We found that HSCs in PU.1-URE^-/-mice were significantly reduced at the myelodysplastic syndrome (MDS) stage, while myeloid progenitors were markedly expanded. Intriguingly, HSCs were distributed in significantly closer proximity to the CD163⁺ subset, which correlated with increased quiescence. These results suggest that the CD163⁺ subset provided a niche for the regulation of HSC cycling. By contrast, myeloid progenitors were localized in close proximity to the CD163^-subset, and the distances were further reduced in PU.1-URE^-/-mice. Collectively, our data suggest that macrophage subsets in the BM provide distinct niches that support diverse hematopoietic processes in steady state and clonal diseases.