Bone Marrow Myeloid Cells Regulate Myeloid-Biased Hematopoietic Stem Cells via a Histamine-Dependent Feedback Loop

Abstract

Myeloid-biased hematopoietic stem cells (MB-HSCs) play critical roles in recovery from injury, but little is known about how they are regulated within the bone marrow niche. Here we describe an auto-/paracrine physiologic circuit that controls quiescence of MB-HSCs and hematopoietic progenitors marked by histidine decarboxylase (Hdc). Committed Hdc⁺ myeloid cells lie in close anatomical proximity to MB-HSCs and produce histamine, which activates the H₂ receptor on MB-HSCs to promote their quiescence and self-renewal. Depleting histamine-producing cells enforces cell cycle entry, induces loss of serial transplant capacity, and sensitizes animals to chemotherapeutic injury. Increasing demand for myeloid cells via lipopolysaccharide (LPS) treatment specifically recruits MB-HSCs and progenitors into the cell cycle; cycling MB-HSCs fail to revert into quiescence in the absence of histamine feedback, leading to their depletion, while an H₂ agonist protects MB-HSCs from depletion after sepsis. Thus, histamine couples lineage-specific physiological demands to intrinsically primed MB-HSCs to enforce homeostasis.

Keywords: H2 receptor; bone marrow niche; hematopoietic stem cells; histamine; histidine decarboxylase; myeloid biased; quiescence; self-renewal.

Figure 1. Hdc Expression Identifies MB-HSC

(A) Percentage of Hdc-GFP^hi cells in lineages of bone marrow (BM) cells (n = 3 - 7 per group). Six independent experiments. (B) mRNA expression of Hdc gene in BM cells and stromal cells (n = 3 - 5). (C) Quantification of Hdc-GFP^hi HSCs in Hdc-GFP and WT mice (n = 3 per group). (D) Hdc mRNA expression in Hdc-GFP^hi and Hdc-GFP^lo BM HSCs and HSPCs (n = 3 per group). (E) Contribution of Hdc-GFP^hi HSCs (n = 12) to lethally irradiated recipients. (F) Blood myeloid/lymphoid ratio of recipients in (E). (G-J) Relative mRNA expression of myeloid (G) and lymphoid lineage (H), cell cycle (I), and quiescence signatures genes (J) in HSCs (n = 3 - 4 per group). Data were analyzed with one-way analysis of variation (ANOVA) with Bonferroni post-hoc test (A and B) or two-tailed Student’s t-test (C-J). See also Figure S1-S3.

Figure 2. Myeloid Stimuli Activates Hdc-GFP^hi MB-HSC

(A) TLR4 mRNA in HSCs (n = 3 - 4) and myeloid cells (n = 5). (B) TLRs expression from gene microarray analysis in myeloid cells (n = 3 per group). (C) Quantification of HSCs, HSPCs, and myeloid cells at 24 hours after LPS treatment (n = 5). (D) Myeloid cells (Gr1⁺) and progenitors (c-kit⁺) in spleen from LPS (n = 4) or PBS (n = 3)-treated Hdc-GFP mice. Four independent experiments. (E and F) Cell cycle analysis of BM HSCs at 6 hours after PBS (n = 3) or LPS (n = 3) treatment. (G) Absolute numbers of HSC in BM of Hdc-GFP mice treated with PBS, IFN-γ, or IL6 (n = 5 per group). Data were analyzed with two-tailed Student’s t-test (A, F, and G) or one-way analysis of variation (ANOVA) with Bonferroni post-hoc test (C). See also Figure S3.

Figure 3. Hdc-GFP^hi MB-HSCs Are Not Responsive to IL-7

(A) Relative mRNA expression of IL-7/IL7-R pathway genes in HSPCs (n = 4 per group). (B) Quantification of Hdc-GFP^hi HSPCs in IL-7 or PBS-treated mice (n = 3 per group). (C) Percentage of G0 HSCs (n = 3 per group). (D) Frequencies of progenitors in IL-7 or PBS-treated Hdc-GFP mice (n = 5 per group). (E) Schematic depiction of the relative lineage bias of Hdc-GFP^hi HSCs or Hdc-GFP^lo HSCs. Data were analyzed with two-tailed Student’s t-test (A-D). See also Figure S3.

Figure 4. Hdc Deficiency Leads to MB-HSC Activation

(A and B) Absolute number of myeloid CFU from 150 BM HSCs (A) and 1 × 10⁵ BM cells (B) (n = 3 per group). Three independent experiments. (C) BrdU incorporation of HSCs and HSPCs (n = 5 per group). (D and E) Hdc deficiency (Hdc^−/−) increased HSC and myeloid lineage progenitors. (F) Competitive transplantation assays of 2 × 105 unfractionated BM cells from Hdc^−/− (n = 12) or WT mice (n = 10). (G) Kaplan-Meier curve depicting survival rates of Hdc^−/− (n = 6) and WT mice (n = 5) after 5-FU treatment. Data were analyzed with two-tailed Student’s t-test (A-C, E, and F) or Logrank test (G). See also Figure S4.

Figure 5. Histamine-producing Myeloid Cells Maintain MB-HSCs

(A and B) Spatial relationship between MB-HSC (a to c, yellow arrow, n = 198) or LB-HSC (d, red arrow, n = 321) and Hdc-GFP⁺ myeloid cells. (C) Percentages of MB-HSCs (n = 541) and random spots (n = 2480) that contacted directly with ≥ 2 Hdc-GFP⁺ myeloid cells. (D) G0 BM HSCs in Hdc-CreER^T2; tdTomato; iDTR (n = 6) and Hdc-CreER^T2_; tdTomato mice (n = 6) as depicted in Figure S5E. Cell cycle rescue was performed by GFP^hiCD11b⁺Gr1⁺ cells transfer (n = 8). Hdc^−/−; Hdc-GFP^hi myeloid cells were used as control (n = 6). (E) Percentage of donor-derived myeloid, T, and B cells in lethally irradiated recipients transplanted with unfractionated BM cells from Hdc-CreER^T2_; tdTomato; iDTR or control mice (n = 5 per group). (F) G0 Hdc-GFP^hi MB-HSCs in Gr1 monoclonal antibody or rat IgG-treated mice (n = 3 per group). (G) Donor chimerism in recipients transplanted with 2 × 105 WT or H₂R^−/− total BM cells along with the same number of CD45.1 BM cells (n = 10 per group). (H and I) Hdc-GFP^hi HSPCs (n = 6) and Hdc-GFP^lo HSPCs (n = 5) co-cultured with Hdc^−/− stromal cells and H₂ antagonist or agonist. Data were analyzed with Mann-Whitney test (B and C), one-way analysis of variation (ANOVA) with Bonferroni post-hoc test (D, H, and I), or two-tailed Student’s t-test (E-G). See also Figure S5.

Figure 6. Myeloid-derived Histamine Protects MB-HSCs from Myelosuppressive Injury

(A) Quantification of Hdc-GFP^hi HSCs in irradiated Hdc-GFP mice (n = 4 - 6 per time point). (B) Number of BM Hdc-GFP⁺ myeloid cells in (A). (C) Competitive reconstitution comparison between 3 Gy-irradiated 1,500 Hdc-GFP^hi and Hdc-GFP^lo HSPCs (n = 5 per group). (D and E) Number of total HSCs (D) or myeloid cells (E) in 5 Gy-irradiated Hdc-GFP (n = 4) and Hdc^−/−; Hdc-GFP mice (n = 6). (F) Blood chimerism of lethally irradiated recipients transplanted with 5 × 10⁵ unfractionated Hdc-GFP⁺ donor BM cells along with Sca-1-depleted CD45.1 BM cells (n = 5 each treatment). (G) Survival of 8Gy- irradiated mice pre-treated with either H₂ agonist or PBS (n = 15 per group). (H) Absolute number of BM Hdc-GFP^hi HSPCs and MPP (MPP3) in LPS or PBS-treated mice at 24 hours (n = 3 - 6 per group). (I) Protective effect of H₂ agonist on LPS-induced sepsis mice (n = 5 per group). Data were analyzed with one-way analysis of variation (ANOVA) with Bonferroni post-hoc test (A, B, and I), two-tailed Student’s t-test (C-E, F, and H), or Logrank test (G). See also Figure S6. For all panels, ± SEM is shown. *p < 0.05; **p < 0.01; ***p < 0.001. n.s., not significant. n.d., not detectable. n indicates biological replicates. For all experiments greater than or equal to two independent experiments were performed unless otherwise indicated.