Murine erythroid short-term radioprotection requires a BMP4-dependent, self-renewing population of stress erythroid progenitors

Abstract

Acute anemic stress induces a systemic response designed to increase oxygen delivery to hypoxic tissues. Increased erythropoiesis is a key component of this response. Recovery from acute anemia relies on stress erythropoiesis, which is distinct from steady-state erythropoiesis. In this study we found that the bone morphogenetic protein 4-dependent (BMP4-dependent) stress erythropoiesis pathway was required and specific for erythroid short-term radioprotection following bone marrow transplantation. BMP4 signaling promoted the development of three populations of stress erythroid progenitors, which expanded in the spleen subsequent to bone marrow transplantation in mice. These progenitors did not correspond to previously identified bone marrow steady-state progenitors. The most immature population of stress progenitors was capable of self renewal while maintaining erythropoiesis without contribution to other lineages when serially transplanted into irradiated secondary and tertiary recipients. These data suggest that during the immediate post-transplant period, the microenvironment of the spleen is altered, which allows donor bone marrow cells to adopt a stress erythropoietic fate and promotes the rapid expansion and differentiation of stress erythroid progenitors. Our results also suggest that stress erythropoiesis may be manipulated through targeting the BMP4 signaling pathway to improve survival after injury.

Figure 1. Mice transplanted with f mutant bone marrow exhibit a defect in erythroid short-term radioprotection.

C57BL/6 mice were irradiated and transplanted with C57BL/6-f/f mutant or C57BL/6 control bone marrow. (A–C) Analysis of mice transplanted with 1 × 10⁵ unfractionated bone marrow cells. (A–C) Survival of mice transplanted with mutant or control bone marrow (A), hematocrit recovery (B), and reticulocyte production (C) following transplant. (D) Analysis of hematocrit recovery in mice transplanted with 5 × 10⁵ unfractionated bone marrow cells. For each figure, each time point represents 4–18 recipients from at least 3 independent experiments. (E) Donor-derived (CD45.2⁺) spleen cells were isolated by FACS on the indicated days from CD45.1 mice that had been transplanted with 5 × 10⁵ mutant or control bone marrow cells. Cells (2 × 10⁶) were plated in methyl­cellulose media containing Epo only. Stress BFU-Es were scored 5 days later. No colonies were observed prior to day 8 following transplant. ND, not detected. *P < 0.05; **P < 0.01; ***P < 0.005.

Figure 2. Analysis of stress erythroid progenitor populations by flow cytometry and colony assays.

Spleen cells were isolated from CD45.1 mice transplanted with mutant (C57BL/6-f/f) or control (C57BL/6) bone marrow cells. (A) Spleen cells from mice transplanted with control bone marrow cells were labeled with anti-Kit, CD71, and Ter119 antibodies, then gated on Kit⁺ cells. Expression of CD71 and Ter119 was analyzed by flow cytometry. Populations I–III are indicated. (B) Total number of each stress progenitor population in the spleen after transplant. Data represent the average of 3 mice. (C) Enlarged representation of boxed region in B. (D) Total number of Sca1⁺ cells in the spleen following transplant. (E) Stress erythroid progenitors in the spleens of mice transplanted with mutant or control bone marrow cells after transplant. Cells were analyzed as described in A. (F) Total number of Populations I–III in mice transplanted with mutant or control bone marrow cells. (G–I) BFU-E colony-forming potential of Population I cells (G) and CFU-E colony-forming potential of Population I (H) and Population II (I) cells isolated by FACS from the spleens of mice transplanted with mutant or control bone marrow cells, on day 8 after transplant. Cells were plated in methylcellulose media containing the indicated growth factors and cultured at 20% or 2% O₂. For all conditions, control cells produced significantly more BFU-Es or CFU-Es than mutant cells (P < 0.05). *P < 0.05; **P < 0.01; ***P < 0.005. For each time point, at least 3 independent mice were analyzed.

Figure 3. Analysis of bone marrow progenitor populations that give rise to BMP4-dependent stress erythroid progenitors in the spleen following transplant.

(A) Stress BFU-Es generated by bone marrow KSL cells on day 8 after transplant. (B) Stress BFU-Es produced by bone marrow CD34⁺ KSLs and CD34^– KSLs on day 8 after transplant.

Figure 4. Scl and Gata2 expression is regulated by BMP4 during the differentiation of stress erythroid progenitors.

(A) qRT-PCR analysis of Scl (left) and Gata2 (right) expression in Population I progenitor cells sorted from mice transplanted with f/f mutant or control bone marrow on days 6 and 8 after transplant. Expression is relative to Gapdh. (B). Population I stress progenitors were sorted on the indicated days after transplant and incubated alone or with BMP4, Shh, SCF, and hypoxia for 2 hours. Scl (left) and Gata2 (right) expression was determined by qRT-PCR. Expression is relative to Gapdh. (C) Rescue of the f/f defect in stress BFU-E development by retroviral expression of Scl or Gata2. f/f mutant bone marrow cells infected with the indicated viruses were transplanted in mice, and on day 8 after transplant, spleen cells were isolated and plated for stress BFU-Es in methylcellulose media containing either Epo alone or Epo, BMP4, and hypoxia.

Figure 5. Population I cells can provide erythroid short-term radio protection when transplanted into secondary recipients.

(A) Population I and II cells isolated by FACS from the spleens of CD45.1 mice transplanted with 5 × 10⁵ control (CD45.2⁺) bone marrow cells on day 8 after transplant were transplanted into lethally irradiated CD45.1 secondary recipients. The secondary transplants were analyzed for survival (B), hematocrit recovery (C), reticulocyte generation (D), rbc count (E), hemoglobin (F), platelet count (G), and wbc count (H) in Population I and II secondary transplants. The values for the recovery of mice transplanted with unfractionated bone marrow transplants and control untreated mice are indicated. (I) Flow cytometry analysis of peripheral blood mononuclear cells. Cells were harvested 42 days after secondary transplant and stained with anti-CD45.2 and the indicated antibodies (top) or isotype controls (bottom). Data are representative of 3 mice. (J) Analysis of Hbb alleles on cellulose acetate gels. Secondary transplants (>48 days after transplant) were treated with PHZ to induce anemia. Hbb alleles were tested 7 days later (1^st PHZ). The mice were allowed to recover 52 days, then were treated a second time with PHZ, and Hbb alleles were analyzed 7 days later (2^nd PHZ). (K). Donor-derived cells were sorted from the spleen or bone marrow of secondary recipients. The cells were plated in “complete” methylcellulose media containing SCF⁺IL-3⁺IL-6⁺ Epo. Colonies were counted and scored as BFU-E or “other” myeloid colonies.

Figure 6. Model for erythroid short-term radioprotection during the recovery from bone marrow transplant.

Details are in the text. HH, hedgehog.