The microbiota regulates hematopoietic stem cell fate decisions by controlling iron availability in bone marrow

Abstract

Host microbiota crosstalk is essential for the production and functional modulation of blood-cell lineages. Whether, and if so how, the microbiota influences hematopoietic stem cells (HSCs) is unclear. Here, we show that the microbiota regulates HSC self-renewal and differentiation under stress conditions by modulating local iron availability in the bone marrow (BM). In microbiota-depleted mice, HSC self-renewal was enhanced during regeneration, while the commitment toward differentiation was dramatically compromised. Mechanistically, microbiota depletion selectively impaired the recycling of red blood cells (RBCs) by BM macrophages, resulting in reduced local iron levels without affecting systemic iron homeostasis. Limiting iron availability in food (in vivo) or in culture (ex vivo), or by CD169⁺ macrophage depletion, enhanced HSC self-renewal and expansion. These results reveal an intricate interplay between the microbiota, macrophages, and iron, and their essential roles in regulating critical HSC fate decisions under stress.

Keywords: erythrophagocytosis; fate decision; hematopoietic regeneration; hematopoietic stem cell; iron; macrophage; microbiota; self-renewal.

Figure 1.. Microbiota depletion leads to impaired HSC response in regenerative condition

(A) Neutrophils in the blood of control and ABX-treated mice after 5FU challenge (n = 6–27). See also Figure S1D. (B–D) Total cellularity, cell lineages, Lin⁻ cells, MPPs and HSCs in the BM of control and ABX-treated mice at day 12 after 5FU challenge (n = 12–32). See also Figure S1F–G for erythroblasts and time-course analysis of HSCs. (E) BMT analysis of control and ABX-treated mice under steady state or at day 12 after 5FU treatment (n = 5–7). See also Figure S1H–I for multi-lineage reconstitution and the HSCT analysis. * p < 0.05, ** p < 0.01, *** p < 0.001. Error bars, mean ± SEM. See also Figure S1 and S2 for steady-state and early time-point analyses and the germ-free model.

Figure 2.. The microbiota regulates HSC response in different stress conditions

(A–C) Cell lineages, Lin⁻ cells, MPPs and HSCs in the BM of control and ABX-treated mice at day 24 after sublethal TBI (n = 4–5). See also Figure S2G. (D–E) BMT and HSCT analyses of control and ABX-treated mice under steady state or at day 24 after sublethal TBI (n = 4–9). (F) HSCs in the blood and BM of control and ABX-treated mice following G-CSF treatment (n = 6–8). (G–I) LSK cells and HSCs in the BM of control and ABX-treated mice following E. coli infection or LPS challenge (n = 3–4). See also Figure S2H–I. * p < 0.05, ** p < 0.01, *** p < 0.001. Error bars, mean ± SEM. See also Figure S3 for niche, HSC localization, and cytokine availability analyses.

Figure 3.. The microbiota regulates erythrophagocytosis and iron availability in the BM during regeneration

(A) Biotin pulse-chase labelling to evaluate RBC clearance in control and ABX-treated mice after 5FU challenge (n = 4–10). (B) Time-course analysis of macrophages in the BM of control and ABX-treated mice after 5FU challenge (n = 6–16). See also Figure S4B and S4C for gene expression analyses and splenic macrophages. (C–D) Adoptive transfer of GFP⁺ RBCs to evaluate RBC clearance and phagocytosis by macrophages in the BM under steady state or in the early phase of regeneration (day 3–5; n = 7–11). See also Figure S4D–G for other tissues and later time points. (E) Iron levels in the BM, spleen and liver of control and ABX-treated mice under steady state or at day 12 after 5FU challenge (n= 8–19). (F) CD71 levels on HSCs in control and ABX-treated mice under steady state or at day 12 after 5FU challenge (n= 18–22). See also Figure S4H–J for germ-free and sublethal irradiation models. * p < 0.05, ** p < 0.01, *** p < 0.001. Error bars, mean ± SEM. See also Figure S4K for systemic iron parameters.

Figure 4.. CD169⁺ BM macrophages control the delivery of iron to HSCs for regeneration

(A) RBCs in the blood of control and CD169-DTR mice after 5FU challenge (n = 9–13). (B–C) BM macrophages and iron levels in the BM and sera of control and CD169-DTR mice under steady state or at day 12 after 5FU challenge (n = 4–15). See also Figure S5A for iron levels in other tissues. (D–F) BM cellularity, neutrophils, Lin⁻ cells, HSCs, and long-term reconstitution following BMT in control and CD169-DTR mice under steady state or at day 12 after 5FU challenge (n = 6–15). See also Figure S5B–E. (G–H) BM iron levels, Lin⁻ cells, and HSCs in control and CD169-DTR mice with or without ABX treatment at day 12 after 5FU challenge (n = 4–10). See also Figure S5F. * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant. Error bars, mean ± SEM.

Figure 5.. The microbiota orchestrates regenerative events in the BM via short-chain fatty acid butyrate

(A–B) BM cellularity, neutrophils, Lin⁻ cells, and HSCs in control, Tlr4^−/−, Tlr2 ^−/− and LysM-Cre/Myd88^fl/fl mice at day 12 after 5FU challenge (n = 5–14). See also Figure S5G–J for additional mouse models and the LPS add-back experiment. (C–E) RBC phagocytosis by bone marrow-derived macrophages ex vivo in the presence of microbial metabolites or HDAC inhibitors (n = 7–9). See also Figure S6A and S6B. (F–G) RBC phagocytosis in control, ABX-treated and ABX-treated mice supplemented with butyrate, in the early phase of regeneration (day 3–5; n = 6–8). See also Figure S6E for gene expression analyses. (H–K) BM iron levels, neutrophils, Lin⁻ cells, HSCs, and long-term reconstitution following BMT in control, ABX-treated and ABX-treated mice supplemented with butyrate, at day 12 after 5FU treatment (n = 6–16). See also Figure S6F–H. * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant. Error bars, mean ± SEM.

Figure 6.. Iron availability regulates HSC self-renewal and differentiation during regeneration

(A–C) Neutrophils, Lin⁻ cells, HSCs, and long-term reconstitution following BMT in mice fed with normal, iron-low or iron-deficient food, at day 12 after 5FU challenge (n= 8–11). See also Figure S7D–G. (D–E) Neutrophils, Lin⁻ cells, HSCs and HSC cell cycling in the BM of ABX-treated mice fed with normal, iron-low or iron-deficient food, at day 12 after 5FU treatment (n = 4–9). (F–H) Neutrophils, Lin⁻ cells, HSCs, and long-term reconstitution following BMT in control and ABX-treated mice injected with PBS or iron dextran, at day 12 after 5FU challenge (n = 5–14). See also Figure S7H–L. * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant. Error bars, mean ± SEM. See also Figure S7 for steady-state analyses of the iron deficiency model.

Figure 7.. Limiting iron availability stimulates HSC self-renewal and expansion ex vivo

(A–B) Total cell and CD150⁺ HSC numbers in the PVA-based culture system with defined transferrin levels (n = 7–8). (C–D) Lin⁻ cell ratio, cell cycling and ROS production of HSCs in the PVA-based culture system with defined transferrin levels (n = 4–8). (E) Transplantation analysis of HSCs expanded with 500 mg/L or 0.5 mg/L transferrin (n = 4–5). (F) Model figure showing a Goldilocks zone for HSC ex vivo expansion, with higher or lower iron levels impairing HSC self-renewal. * p < 0.05, ** p < 0.01, *** p < 0.001. Error bars, mean ± SEM.