Extramedullary erythropoiesis in the adult liver requires BMP-4/Smad5-dependent signaling

Abstract

Objective: In mice, homeostatic erythropoiesis occurs primarily in the bone marrow. However, in response to acute anemia, bone morphogenetic proteins 4 (BMP-4)-dependent stress erythropoiesis occurs in the adult spleen. BMP-4 can also regulate stress erythropoiesis in the fetal liver. In humans, erythropoiesis occurs in the bone marrow. However, in certain pathological conditions, extramedullary erythropoiesis is observed, where it can occur in several organs, including the liver. Given these observations, we propose to investigate whether the BMP-4-dependent stress erythropoiesis pathway can regulate extramedullary erythropoiesis in the livers of splenectomized mice.

Materials and methods: Using splenectomized wild-type and flexed-tail (f) mice, which have a defect in BMP-4 signaling, we compared their recovery from phenylhydrazine-induced hemolytic anemia and characterized the expansion of stress burst-forming unit-erythroid in the livers of these mice during the recovery period.

Results: Our analysis indicates that in the absence of a spleen, stress erythropoiesis occurs in the murine liver. During the recovery, stress burst-forming unit-erythroid are expanded in the livers of splenectomized mice in response to BMP-4 expressed in the liver. f/f mice, which exhibit a defect in splenic stress erythropoiesis do not compensate for this defect by upregulating liver stress erythropoiesis. Furthermore, splenectomized f/f mice exhibit a defect in liver stress erythropoiesis, which demonstrates a role for the BMP-4-dependent stress erythropoiesis pathway in extramedullary erythropoiesis in the adult liver.

Conclusions: Our data indicate that the BMP-4-dependent stress erythropoiesis pathway regulates extramedullary stress erythropoiesis, which occurs primarily in the murine spleen or in the case of splenectomized mice, in the adult liver.

Figure 1. Recovery from PHZ induced acute hemolytic anemia

Splenectomized C57BL/6 and C57BL/6 control mice were treated with PHZ to induce anemia. Hematocrit measurements were done at the indicated times. The data shown are the average ± standard deviation of at least 3 mice per time point. *p<0.05, **p<0.01.

Figure 2. Analysis of BFU-E in the bone marrow and liver during the recovery from PHZ induced anemia

(A). Analysis of BFU-E in the bone marrow during the recovery from acute anemia. Bone marrow cells were plated in methylcellulose media supplemented with Epo (3U/ml) and IL-3 (50ng/ml). The data are the number of BFU-E per 1×10⁵ bone marrow cells. (B) Analysis of BFU-E in the liver during recovery from acute anemia. Liver mononuclear cells (1×10⁴ per well) were plated in methylcellulose media supplemented with Epo + IL-3 (left) or Epo alone (right). Statistically significant differences are indicated on the figure. The figure shows a representative experiment of 2 independent experiments. The data shown are the average ± standard deviation of at least 3 mice per time point.

Figure 3. Analysis of BMP4 expression in the liver during recovery from acute anemia

(A) Hematoxylin and eosin stained liver sections from C57BL/6-splenectomized and C57BL/6 control mice (Top) and C57BL/6-f/f splenectomized and C57BL/6-f/f control mice (bottom) at indicated time points following a PHZ induced acute anemia. Examples of the darker staining erythropoietic clusters are shown in gray boxed area marked in the day 6 in C57BL/6 splenectomized and control mice and on day 8 in the C57BL/6-f/f splenectomized and control mice. Magnified views of the gray boxed regions are shown to the right of the panels. At least two independent livers were analyzed at each time point. Each liver was fixed, cut into several pieces and sectioned so that each section had fields from multiple areas of the liver. 2–3 sections per time point were analyzed and representative fields shown. (B) Schematic of liver acinus (functional interpretation of liver organization) formed from classical liver lobules. Blood enters the liver from portal triads composed of portal vein, hepatic artery and bile duct flows towards the central vein. This creates a gradient of oxygen concentration ranging highest in zone 1 (1), intermediate in zone 2 (2), to lowest in zone 3 (3). (C) Liver sections from C57BL/6-splenectomized and C57BL/6 control mice and (D) C57BL/6-f/f splenectomized and C57BL/6-f/f control mice recovering from an acute anemia stained with anti-BMP4 antibodies and HRP-conjugated secondary antibodies (BMP4) or secondary alone (control). At least two independent livers were analyzed at each time point. Each liver was fixed, cut into several pieces and sectioned so that each section had fields from multiple areas of the liver. 2–3 sections per time point were analyzed and representative fields shown. (*) denotes portal vein of triad composed of portal vein, hepatic artery and bile duct. Arrow points to a central vein. BMP4 staining is brown, with blue hematoxylin counter-stain. Note an example of how BMP4 expression remains expanded into zones 1 and 2 (bracket) into Day 6 post-PHZ in splenectomized mice, while it returns to exclusively zone 3 (3) in wild-type mice by 4 Days post anemia.

Figure 3. Analysis of BMP4 expression in the liver during recovery from acute anemia

(A) Hematoxylin and eosin stained liver sections from C57BL/6-splenectomized and C57BL/6 control mice (Top) and C57BL/6-f/f splenectomized and C57BL/6-f/f control mice (bottom) at indicated time points following a PHZ induced acute anemia. Examples of the darker staining erythropoietic clusters are shown in gray boxed area marked in the day 6 in C57BL/6 splenectomized and control mice and on day 8 in the C57BL/6-f/f splenectomized and control mice. Magnified views of the gray boxed regions are shown to the right of the panels. At least two independent livers were analyzed at each time point. Each liver was fixed, cut into several pieces and sectioned so that each section had fields from multiple areas of the liver. 2–3 sections per time point were analyzed and representative fields shown. (B) Schematic of liver acinus (functional interpretation of liver organization) formed from classical liver lobules. Blood enters the liver from portal triads composed of portal vein, hepatic artery and bile duct flows towards the central vein. This creates a gradient of oxygen concentration ranging highest in zone 1 (1), intermediate in zone 2 (2), to lowest in zone 3 (3). (C) Liver sections from C57BL/6-splenectomized and C57BL/6 control mice and (D) C57BL/6-f/f splenectomized and C57BL/6-f/f control mice recovering from an acute anemia stained with anti-BMP4 antibodies and HRP-conjugated secondary antibodies (BMP4) or secondary alone (control). At least two independent livers were analyzed at each time point. Each liver was fixed, cut into several pieces and sectioned so that each section had fields from multiple areas of the liver. 2–3 sections per time point were analyzed and representative fields shown. (*) denotes portal vein of triad composed of portal vein, hepatic artery and bile duct. Arrow points to a central vein. BMP4 staining is brown, with blue hematoxylin counter-stain. Note an example of how BMP4 expression remains expanded into zones 1 and 2 (bracket) into Day 6 post-PHZ in splenectomized mice, while it returns to exclusively zone 3 (3) in wild-type mice by 4 Days post anemia.

Figure 4. Recovery of splenectomized f/f mice from acute anemia is compromised

(A). Splenectomized C57BL/6-f/f and C57BL/6 control mice were treated with PHZ to induce acute anemia. Hematocrit was measured on the indicated days. *p<0.05, **p<0.01, ***p<0.001. (B) Survival curve of splenectomized C57BL/6-f/f and C57BL/6 control mice were treated with PHZ. Recovery from PHZ induced anemia was done in 2 independent experiments. 4–6 mice were used per time point. (C) Analysis of BFU-E in the liver of splenectomized C57BL/6-f/f and C57BL/6 control mice were treated with PHZ. Liver mononuclear cells (1×10⁴ per well) were plated in methylcellulose media supplemented with Epo + IL-3 (left) or Epo alone (right). Statistically significant differences are indicated on the figure. The figure shows average ± standard deviation of four mice per time point.