G-CSF shifts erythropoiesis from bone marrow into spleen in the setting of systemic inflammation

G-CSF suppresses bone marrow erythropoiesis in mice in a dose- and time-dependent and reversible manner

To characterize the effect of G-CSF on erythropoiesis globally, we first performed a detailed analysis of the relationship between G-CSF dosage and alteration in erythropoiesis in the bone marrow of mice. G-CSF (1, 2, 5, 10, 20, 50, or 100 μg/kg) was subcutaneously administered twice daily to male C57BL/6 mice for 9 d. The mouse femurs became progressively paler in response to the gradually increasing doses of G-CSF (Fig 1A). Furthermore, cell-counting analysis revealed a gradual reduction in bone marrow cell number after G-CSF treatment in a dose-dependent fashion (Fig 1B). These observations suggest G-CSF treatment causes a profound alteration of hematopoiesis in bone marrow. Therefore, we performed flow cytometry to examine erythroid differentiation in the bone marrow of femurs using two markers of erythroid lineage, Ter119 and CD71. We found that the percentage of Ter119⁺CD71⁺ cells in the bone marrow of mice gradually decreased with increasing doses of G-CSF (Fig 1C and D). Thiazole orange is a fluorescent dye that is cell membrane permeable and binds to DNA and RNA (30). Reticulocyte contains a small amount of RNA and DNA. Ter119 is a cell-surface erythroid-specific marker and is expressed from proerythroblast to mature erythrocyte (13). Thiazole orange and Ter119-APC double staining can characterize the reticulocytes (Ter119⁺Thiazole orange⁺) and erythroblasts (Ter119⁺Thiazole orange⁺⁺) in the bone marrow. We next carried out flow cytometric analysis by double staining with thiazole orange and Ter119-APC to further characterize the red blood cell precursors in G-CSF-treated mice. After treatment of mice with G-CSF, the numbers of reticulocytes and erythroblasts in the bone marrow gradually decreased (Fig 1E–G). Burst-forming unit erythroid (BFU-E) is the earliest committed erythroid progenitor (31). We found that bone marrow cells from G-CSF–treated mice (50 μg/kg) formed lower numbers of BFU-E colonies than the controls (Fig 1H and I). GATA-1, which promotes erythroid differentiation by regulating the expression of several erythroid-specific genes, is highly expressed during erythroid differentiation (32, 33). To further understand the link between G-CSF and erythropoiesis, we studied changes in GATA-1 expression in bone marrow cells in mice treated with G-CSF (50 μg/kg) using flow cytometry with specific antibodies. GATA-1 expression was decreased in the bone marrow cells of mice treated with G-CSF (Fig 1J and K).

• Download figure
• Open in new tab
• Download powerpoint

Figure 1. BM erythropoiesis is gradually inhibited in G-CSF–treated mice.

(A) Representative images of femurs from control and G-CSF–treated mice (scale bar = 1 cm). (B) Quantification of total BM cells. (C) Representative flow cytometric plots of CD71⁺Ter119⁺ cells in the BMs of control and G-CSF–treated mice. (D) Flow cytometric quantification of CD71⁺Ter119⁺ erythroid cells in the BMs of control and G-CSF-treated mice. (E) Representative flow cytometric profiles of Ter119-APC and thiazole orange-stained cells from the BMs of control and G-CSF–treated mice. (F) Flow cytometric quantification of reticulocytes (Ter119⁺ Thiazole orange⁺) in the BM of mice. (G) Flow cytometric quantification of erythroblasts (Ter119⁺ Thiazole orange⁺⁺) in the BMs of mice. (H) Representative fields of burst-forming unit erythroid-derived colonies in BM cultures from control and G-CSF–treated mice (×100 magnification; scale bar = 200 μm). (I) Quantification of the number of burst-forming unit erythroid-derived colonies in BM cultures from control and G-CSF treated mice. (J, K) Flow cytometric analyses of GATA-1 expression in BM cells from control and G-CSF–treated mice. (J) Representative histograms. (K) Mean fluorescence intensities were graphed. (L) Representative images of femurs from control and G-CSF–treated mice (scale bar = 1 cm). (M) Quantification of total BM cells. (N) Representative flow cytometric plots of CD71⁺Ter119⁺ BM cells for each indicated group. (O) Flow cytometric quantification of CD71⁺Ter119⁺ erythroid cells in the BMs of control and G-CSF–treated mice. (P) Representative flow cytometric profiles of BM cells stained with anti-Ter119 and thiazole orange for each indicated group. (Q) Flow cytometric quantification of reticulocytes (Ter119⁺ Thiazole orange⁺) in the BM of mice. (R) Flow cytometric quantification of erythroblasts (Ter119⁺ Thiazole orange⁺⁺) in the BMs of mice. n = 5/group (A, B), n = 3/group (C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R). **P < 0.01, ***P < 0.001 versus controls. Data are from one experiment representative of three experiments.

Next, we evaluated erythropoiesis suppression by G-CSF in the bone marrow over time. Bone marrow erythropoiesis began to be significantly inhibited at 24 h after the first injection of G-CSF (50 μg/kg) and continued through G-CSF administration, as indicated by the pale bone color, decreased number of bone marrow cells, and decreased proportion of Ter119⁺CD71⁺ cells, reticulocytes, and erythroblasts (Fig 1L–R). We next explored whether depression of bone marrow erythropoiesis by G-CSF is reversible. Flow cytometric analyses showed that the Ter119⁺CD71⁺ cell, reticulocyte, and erythroblast populations in the bone marrow were restored after G-CSF withdrawal for 15 d and the bone color and bone marrow cell number also recovered after stopping treatment of mice with G-CSF (Fig S1A–G). We also treated mice with single dose of G-CSF (50 μg/kg), and we found that suppression of bone marrow erythropoiesis occurred in mice exposed to single dose of G-CSF (Fig S2A–G). Together, these observations suggest G-CSF reversibly suppresses erythropoiesis in the bone marrow in a dose- and time-dependent manner.

• Download figure
• Open in new tab
• Download powerpoint

Figure S1. G-CSF withdrawal restores BM erythropoiesis and decreases splenic erythropoiesis in G-CSF–treated mice.

G-CSF was administered to male C57BL/6 mice at 50 μg/kg for 9 d by twice-daily subcutaneous injections, and then was stopped for 3, 6, 9, 12, and 15 d. (A) Representative images of femurs from normal control mice, G-CSF–treated mice, and stopping treatment of mice with G-CSF (scale bar = 1 cm). (B) Quantification of total BM cells. (C) Representative flow cytometric plots of CD71⁺Ter119⁺ cells from the BMs of normal control mice, G-CSF–treated mice, and stopping treatment of mice with G-CSF. (D) Flow cytometric quantification of CD71⁺Ter119⁺ erythroid cells in the BMs of normal control mice, G-CSF–treated mice, and stopping treatment of mice with G-CSF. (E) Representative flow cytometric profiles of Ter119-APC and thiazole orange–stained cells from the BMs of control mice, G-CSF–treated mice, and stopping treatment of mice with G-CSF. (F) Flow cytometric quantification of reticulocytes (Ter119⁺ Thiazole orange⁺) in the BM of mice. (G) Flow cytometric quantification of erythroblasts (Ter119⁺ Thiazole orange⁺⁺) in the BMs of mice. (H) Representative flow cytometric plots of CD71⁺Ter119⁺ cells in the spleens of control mice, G-CSF–treated mice, and stopping treatment of mice with G-CSF. (I) Bar graphs showing quantification of CD71⁺Ter119⁺ cells in the spleens of control mice, G-CSF–treated mice, and stopping treatment of mice with G-CSF. (J) Representative flow cytometric plots of Ter119-APC and thiazole orange–stained splenocytes from control mice, G-CSF–treated mice, and stopping treatment of mice with G-CSF. (K) Flow cytometric quantification of reticulocytes (Ter119⁺ Thiazole orange⁺) in the spleens from different treatment groups. (L) Flow cytometric quantification of erythroblasts (Ter119⁺ Thiazole orange⁺⁺) in the spleens from different treatment groups. n = 3/group (A, B, C, D, E, F, G, H, I, J, K, L). *P < 0.05, **P < 0.01, ***P < 0.001 versus controls, n.s., no significance.

• Download figure
• Open in new tab
• Download powerpoint

Figure S2. The effects of single dose of G-CSF (50 μg/kg) on BM and spleen.

(A) Representative images of femurs from control and G-CSF–treated mice (scale bar = 1 cm). (B) Quantification of total BM cells. (C) Representative flow cytometric plots of CD71⁺Ter119⁺ cells in the BMs of control and G-CSF–treated mice. (D) Flow cytometric quantification of CD71⁺Ter119⁺ erythroid cells in the BMs of control and G-CSF–treated mice. (E) Representative flow cytometric profiles of Ter119-APC and thiazole orange–stained cells from the BMs of control and G-CSF–treated mice. (F) Flow cytometric quantification of reticulocytes (Ter119⁺ Thiazole orange⁺) in the BM of mice. (G) Flow cytometric quantification of erythroblasts (Ter119⁺ Thiazole orange⁺⁺) in the BMs of mice. (H) Representative flow cytometric plots of CD11b⁺Ly6G⁺ cells in the BMs of control and G-CSF–treated mice. (I) Flow cytometric quantification of CD11b⁺Ly6G⁺ neutrophils in the BMs of control and G-CSF–treated mice. (J) Flow cytometric quantification of CD11b⁺ neutrophils in the BMs of control and G-CSF–treated mice. (K) Representative flow cytometric plots of CD71⁺Ter119⁺ cells in the spleens of control and G-CSF–treated mice. (L) Bar graphs showing quantification of CD71⁺Ter119⁺ cells in the spleens of control and G-CSF–treated mice. (M) Representative flow cytometric plots of Ter119-APC and thiazole orange–stained splenocytes from control and G-CSF–treated mice. (N) Flow cytometric quantification of reticulocytes (Ter119⁺ Thiazole orange⁺) in the spleens from different treatment groups. (O) Flow cytometric quantification of erythroblasts (Ter119⁺ Thiazole orange⁺⁺) in the spleens from different treatment groups. (P) Macroscopic views of spleens in control and G-CSF–treated mice (scale bar = 1 cm). (Q) Average weights of spleens in control and G-CSF–treated mice. (R) Total cell numbers of spleens in control and G-CSF–treated mice. (S) Compressive strength of spleens from each group. n = 3/group (A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, S). *P < 0.05, **P < 0.01, ***P < 0.001.

G-CSF regulates neutrophil production and mobilization from the bone marrow. Therefore, we measured the neutrophil population in the bone marrow of G-CSF–treated mice using flow cytometry and found that the percentage of CD11b⁺Ly6G⁺ cells in the bone marrow gradually increased in response to increasing doses of G-CSF (Fig S3A and B). Analysis of hematological parameters showed G-CSF treatment led to more peripheral blood neutrophils in mice in a dose-dependent manner (Fig S3C). We also assessed blood and bone marrow smears after Wright–Giemsa staining by microscopy. Mice treated with G-CSF (50 μg/kg) had more neutrophils in the bone marrow and peripheral blood than the controls (Fig S3D and E). Quantification of CFU-GM showed that the numbers of CFU-GM derived from the bone marrow of G-CSF–treated mice were increased (Fig S3F and G). Administration of a single dose of G-CSF (50 μg/kg) led to an increase in the levels of CD11b⁺ cells, indicating that one dose of G-CSF initiates the granulopoiesis of bone marrow in mice (Fig S2H–J). These data suggest that G-CSF has different effects on erythropoiesis and granulopoiesis in the bone marrow of mice.

• Download figure
• Open in new tab
• Download powerpoint

Figure S3. G-CSF treatment enhances granulopoiesis in the BM of mice.

(A, B) Flow cytometric analysis of the BM neutrophils (CD11b⁺Ly6G⁺) in control and G-CSF–treated mice. (A) Representative flow cytometric plots of CD11b⁺Ly6G⁺ cells in the BMs of control and G-CSF–treated mice. (B) Flow cytometric quantification of CD11b⁺Ly6G⁺ neutrophils in the BMs of control and G-CSF–treated mice. (C) Peripheral neutrophil counts in control and G-CSF–treated mice. (D) Wright–Giemsa staining of BM cells from control and G-CSF–treated mice. Original magnification ×100 (upper panels); ×1,000 (lower panels) (E) Wright–Giemsa staining of peripheral blood cells from control and G-CSF–treated mice. (F) Representative fields of CFU-GM–derived colonies in BM cultures from control and G-CSF–treated mice (×100 magnification; scale bar = 200 μm). (G) Quantification of the number of CFU-GM–derived colonies in BM cultures from control and G-CSF treated mice. n = 5/group (A, B, C, D, E), n = 3/group (F, G).***P < 0.001.

G-CSF promotes splenic erythropoiesis of mice in a dose- and time-responsive fashion

Splenic erythropoiesis can compensate for the repression of bone marrow erythropoiesis during stress (34). To assess the dose-responsive and time-dependent relationships between G-CSF and splenic erythropoiesis, we performed flow cytometric analysis using Ter119-APC and CD71-PE double staining as well as thiazole orange and Ter119-APC double staining. In sharp contrast to bone marrow, the spleen displayed a marked expansion of erythropoiesis in mice treated with G-CSF in a dose- and time-dependent manner, as indicated by the gradually increasing proportions of Ter119⁺CD71⁺ cells, reticulocytes, and erythroblasts (Fig 2A–J). The numbers of BFU-E colonies were also profoundly increased in the spleen of G-CSF-treated (50 μg/kg) mice (Fig 2K and L), confirming that G-CSF treatment promotes splenic erythropoiesis in mice. We also analyzed expression of GATA-1 in splenocytes by flow cytometry and found an increase in mice treated with G-CSF (50 μg/kg) (Fig 2M and N). This expansion of splenic erythropoiesis was maintained throughout G-CSF administration and gradually contracted following recovery of bone marrow erythropoiesis after terminating G-CSF administration (Fig S1H–L). Single-dose administration of G-CSF (50 μg/kg) was not able to trigger the splenic erythropoiesis (Fig S2K–O). To further confirm the crucial role of splenic erythropoiesis in mice treated with G-CSF, we performed splenectomies in mice, treated the mice with G-CSF (50 μg/kg) for 15 d, and then analyzed the hematologic parameters in peripheral blood. The G-CSF–treated mice that underwent a sham operation developed mild anemia with low red blood cell counts, hemoglobin concentrations, and hematocrits, and increased reticulocyte counts. G-CSF treatment significantly reduced the red blood cell counts, hemoglobin concentrations, hematocrits, and reticulocyte counts in mice that had undergone splenectomies (Fig 2O), indicating that splenic erythropoiesis is indispensable for maintaining red blood cell homeostasis in G-CSF–treated mice.

• Download figure
• Open in new tab
• Download powerpoint

Figure 2. G-CSF enhances splenic erythropoiesis in a dose- and time-dependent manner.

(A) Representative flow cytometric plots of CD71⁺Ter119⁺ cells in the spleens of control and G-CSF–treated mice. (B) Bar graphs showing quantification of CD71⁺Ter119⁺ cells in the spleens of control and G-CSF–treated mice. (C) Representative flow cytometric plots of Ter119-APC and thiazole orange-stained splenocytes from control and G-CSF–treated mice. (D) Flow cytometric quantification of reticulocytes (Ter119⁺ Thiazole orange⁺) in the spleens from different treatment groups. (E) Flow cytometric quantification of erythroblasts (Ter119⁺ Thiazole orange⁺⁺) in the spleens from different treatment groups. (F) Representative flow cytometric plots of CD71⁺Ter119⁺ cells in the spleens of control and G-CSF–treated mice. (G) Bar graphs showing quantification of CD71⁺Ter119⁺ cells in the spleens of control and mice treated with G-CSF at different time points. (H) Representative flow cytometric profiles of splenocytes stained with anti-Ter119 and thiazole orange for each indicated group. (I) Flow cytometric quantification of reticulocytes (Ter119⁺ Thiazole orange⁺) in the spleens from different treatment groups. (J) Flow cytometric quantification of erythroblasts (Ter119⁺ Thiazole orange⁺⁺) in the spleens from different treatment groups. (K) Representative fields of burst-forming unit-erythroid-derived colonies in cultures of splenocytes from control and G-CSF-treated mice (×100 magnification; scale bar = 200 μm). (L) Quantification of burst-forming unit erythroid-derived colonies in cultures of splenocytes from control and G-CSF–treated mice. (M) GATA-1 expression of splenocyte was measured by flow cytometry, and the representative histograms were shown. (N) Bar graphs showing mean fluorescence intensities. (O) Peripheral blood hematologic parameters of sham-operated mice, splenectomized mice, sham-operated mice treated with G-CSF, and splenectomized mice treated with G-CSF. Red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), and reticulocytes (Retics). n = 3/group (A, B, C, D, E, F, G, H, I, J, K, L, M, N), n = 10/group (O). *P < 0.05, **P < 0.01, ***P < 0.001 versus controls. Data are from one experiment representative of three experiments.

Systemic inflammation up-regulates G-CSF levels, inhibits bone marrow erythropoiesis, and promotes splenic erythropoiesis in mice

To examine inflammation-driven dysfunction of erythropoiesis, we used Escherichia coli (E. coli) and LPS to induce systemic inflammation separately. Both systemic infection of mice with E. coli and treatment of mice with LPS resulted in regression of bone marrow erythropoiesis and enhancement of splenic erythropoiesis (Figs 3A–L and S4A–L). The E. coli–infected mice and LPS-treated mice were both phenotypically anemic with low red blood cell counts, hemoglobin concentrations, and hematocrits, and high-reticulocyte counts (Figs 3M and S4M). Accumulating evidence shows G-CSF shapes host immunity by enhancing the production and function of neutrophils in response to infection-related inflammatory stress. Therefore, we determined whether G-CSF is associated with an alteration in erythropoiesis in mice during inflammatory processes. For this purpose, the serum G-CSF levels were analyzed in mice infected with E. coli or treated with LPS. Both systemic E. coli infection and LPS treatment increased serum levels of G-CSF (Figs 3N and S4N). We also measured the populations of neutrophils in the bone marrow and peripheral blood of E. coli–infected and LPS-treated mice and found the numbers of neutrophils (CD11b⁺Ly6G⁺) in both the bone marrow and peripheral blood increased in response to E. coli infection and LPS stimulation (Figs 3O–Q and S4O–Q), indicating that LPS stimulates the secretion of G-CSF and initiates emergency granulopoiesis in bone marrow during systemic inflammation. Single treatment of LPS and single infection of E. coli were associated with regression of bone marrow erythropoiesis, but did not trigger emergency granulopoiesis of bone marrow and splenic erythropoiesis (Figs S5A–N and S6A–N).

• Download figure
• Open in new tab
• Download powerpoint

Figure 3. Infection of mice with E. coli impairs BM erythropoiesis, enhances splenic erythropoiesis, and promotes BM granulopoiesis.

(A) Representative images of femurs from control mice and E. coli–infected mice (scale bar = 1 cm). (B) Quantification of total BM cells. (C) Representative flow cytometric plots of CD71⁺Ter119⁺ cells in the BMs of control mice and E. coli–infected mice. (D) Flow cytometric quantification of CD71⁺Ter119⁺ erythroid cells in the BMs of control and E. coli-infected mice. (E) Representative flow cytometric profiles of Ter119-APC and thiazole orange-stained cells from the BM of control mice and E. coli–infected mice. (F) Flow cytometric quantification of reticulocytes (Ter119⁺Thiazole orange⁺) in the BM of mice. (G) Flow cytometric quantification of erythroblasts (Ter119⁺ Thiazole orange⁺⁺) in the BMs of mice. (H) Representative flow cytometric plots of CD71⁺Ter119⁺ cells in the spleens of control and E. coli-infected mice. (I) Bar graphs showing quantification of CD71⁺Ter119⁺ cells in the spleens of control and E. coli–infected mice. (J) Representative flow cytometric plots of Ter119-APC and thiazole orange–stained splenocytes from control and E. coli–infected mice. (K) Flow cytometric quantification of reticulocytes (Ter119⁺ Thiazole orange⁺) in the spleens from control and E. coli–infected mice. (L) Flow cytometric quantification of erythroblasts (Ter119⁺ Thiazole orange⁺⁺) in the spleens from control and E. coli-infected mice. (M) Peripheral blood hematologic parameters of control mice and E. coli-infected mice. Red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), and reticulocytes (Retics). (N) Serum concentrations of G-CSF in control and E. coli–infected mice. (O) Representative flow cytometric plots of CD11b⁺Ly6G⁺ cells in the BMs of control and E. coli-infected mice. (P) Flow cytometric quantification of CD11b⁺Ly6G⁺ neutrophils in the BMs of control and E. coli–infected mice. (Q) Peripheral neutrophil counts in control and E. coli–infected mice. n = 5/group (A, B, M, Q), n = 3/group (C, D, E, F, G, H, I, J, K, L, N, O, P). **P < 0.01, ***P < 0.001.

• Download figure
• Open in new tab
• Download powerpoint

Figure S4. Treatment of mice with LPS impairs BM erythropoiesis, enhances splenic erythropoiesis, and promotes BM granulopoiesis.

(A) Representative images of femurs from control and LPS-treated mice (scale bar = 1 cm). (B) Quantification of total BM cells. (C) Representative flow cytometric plots of CD71⁺Ter119⁺ cells in the BMs of control and LPS-treated mice. (D) Flow cytometric quantification of CD71⁺Ter119⁺ erythroid cells in the BMs of control and LPS-treated mice. (E) Representative flow cytometric profiles of Ter119-APC and thiazole orange-stained cells from the BM of control and LPS-treated mice. (F) Flow cytometric quantification of reticulocytes (Ter119⁺ Thiazole orange⁺) in the BM of mice. (G) Flow cytometric quantification of erythroblasts (Ter119⁺ Thiazole orange⁺⁺) in the BMs of mice. (H) Representative flow cytometric plots of CD71⁺Ter119⁺ cells in the spleens of control and LPS-treated mice. (I) Bar graphs showing quantification of CD71⁺Ter119⁺ cells in the spleens of control and LPS-treated mice. (J) Representative flow cytometric profiles of Ter119-APC and thiazole orange-stained cells from the spleen of control and LPS-treated mice. (K) Flow cytometric quantification of reticulocytes (Ter119⁺ Thiazole orange⁺) in the spleens from control and LPS-treated mice. (L) Flow cytometric quantification of erythroblasts (Ter119⁺ Thiazole orange⁺⁺) in the spleens from control and LPS-treated mice. (M) Peripheral blood hematologic parameters of control and LPS-treated mice. Red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), and reticulocytes (Retics). (N) Serum concentrations of G-CSF in control and LPS-treated mice. (O) Representative flow cytometric plots of CD11b⁺Ly6G⁺ cells in the BMs of control and LPS-treated mice. (P) Flow cytometric quantification of CD11b⁺Ly6G⁺ neutrophils in the BMs of control and LPS-treated mice. (Q) Peripheral neutrophil counts in control and LPS-treated mice. n = 5/group (A, B, M, Q), n = 3/group (C, D, E, F, G, H, I, J, K, L, N, O, P). **P < 0.01, ***P < 0.001.

• Download figure
• Open in new tab
• Download powerpoint

Figure S5. The effects of single treatment of LPS (0.5 mg/kg) on BM and spleen.

(A) Representative images of femurs from control and LPS-treated mice (scale bar = 1 cm). (B) Quantification of total BM cells. (C) Representative flow cytometric plots of CD71⁺Ter119⁺ cells in the BMs of control and LPS-treated mice. (D) Flow cytometric quantification of CD71⁺Ter119⁺ erythroid cells in the BMs of control and LPS-treated mice. (E) Representative flow cytometric profiles of Ter119-APC and thiazole orange-stained cells from the BMs of control and LPS-treated mice. (F) Flow cytometric quantification of reticulocytes (Ter119⁺ Thiazole orange⁺) in the BM of mice. (G) Flow cytometric quantification of erythroblasts (Ter119⁺ Thiazole orange⁺⁺) in the BMs of mice. (H) Representative flow cytometric plots of CD11b⁺Ly6G⁺ cells in the BMs of control and LPS-treated mice. (I) Flow cytometric quantification of CD11b⁺Ly6G⁺ neutrophils in the BMs of control and LPS-treated mice. (J) Representative flow cytometric plots of CD71⁺Ter119⁺ cells in the spleens of control and LPS-treated mice. (K) Bar graphs showing quantification of CD71⁺Ter119⁺ cells in the spleens of control and LPS-treated mice. (L) Representative flow cytometric plots of Ter119-APC and thiazole orange-stained splenocytes from control and LPS-treated mice. (M) Flow cytometric quantification of reticulocytes (Ter119⁺ Thiazole orange⁺) in the spleens from different treatment groups. (N) Flow cytometric quantification of erythroblasts (Ter119⁺ Thiazole orange⁺⁺) in the spleens from different treatment groups. (O) Macroscopic views of spleens in control and LPS-treated mice (scale bar = 1 cm). (P) Average weights of spleens in control and LPS-treated mice. (Q) Total cell numbers of spleens in control and LPS-treated mice. (R) Compressive strength of spleens from each group. n = 3/group (A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R). **P < 0.01, ***P < 0.001.

• Download figure
• Open in new tab
• Download powerpoint

Figure S6. The effects of single infection of E. coli (1 × 106) on BM and spleen.

(A) Representative images of femurs from control and E. coli–infected mice (scale bar = 1 cm). (B) Quantification of total BM cells. (C) Representative flow cytometric plots of CD71⁺Ter119⁺ cells in the BMs of control and E. coli–infected mice. (D) Flow cytometric quantification of CD71⁺Ter119⁺ erythroid cells in the BMs of control and E. coli–infected mice. (E) Representative flow cytometric profiles of Ter119-APC and thiazole orange–stained cells from the BMs of control and E. coli–infected mice. (F) Flow cytometric quantification of reticulocytes (Ter119⁺ Thiazole orange⁺) in the BM of mice. (G) Flow cytometric quantification of erythroblasts (Ter119⁺ Thiazole orange⁺⁺) in the BMs of mice. (H) Representative flow cytometric plots of CD11b⁺Ly6G⁺ cells in the BMs of control and E. coli-infected mice. (I) Flow cytometric quantification of CD11b⁺Ly6G⁺ neutrophils in the BMs of control and E. coli–infected mice. (J) Representative flow cytometric plots of CD71⁺Ter119⁺ cells in the spleens of control and E. coli–infected mice. (K) Bar graphs showing quantification of CD71⁺Ter119⁺ cells in the spleens of control and E. coli–infected mice. (L) Representative flow cytometric plots of Ter119-APC and thiazole orange–stained splenocytes from control and E. coli–infected mice. (M) Flow cytometric quantification of reticulocytes (Ter119⁺ Thiazole orange⁺) in the spleens from different treatment groups. (N) Flow cytometric quantification of erythroblasts (Ter119⁺ Thiazole orange⁺⁺) in the spleens from different treatment groups. (O) Macroscopic views of spleens in control and E. coli–infected mice (scale bar = 1 cm). (P) Average weights of spleens in control and E. coli–infected mice. (Q) Total cell numbers of spleens in control and E. coli-infected mice. (R) Compressive strength of spleens from each group. n = 3/group (A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R). *P < 0.05, **P < 0.01, ***P < 0.001.

G-CSF neutralization reverses the functional changes of erythropoiesis in the bone marrow and spleens of LPS-treated mice

We then hypothesized that G-CSF impairs bone marrow erythropoiesis and promotes splenic erythropoiesis in mice during LPS-induced inflammation. To test this hypothesis, we examined erythropoiesis in the bone marrow and spleens in the setting of antibody-mediated neutralization of G-CSF. Anti–G-CSF treatment reduced serum G-CSF concentrations in LPS-treated mice (Fig 4A), restored erythropoiesis in the bone marrow (Figs 4B–H and S7A–G), and reversed LPS-induced splenic erythropoiesis (Figs 4I–M and S7H–L), indicating that systemic inflammation influences erythropoiesis in the body by increasing G-CSF production. As expected, G-CSF neutralization blocked emergency granulopoiesis in the bone marrow (Figs 4N and O and S7M and N) and decreased blood neutrophil counts (Fig 4P).

• Download figure
• Open in new tab
• Download powerpoint

Figure 4. G-CSF neutralization decreases BM granulopoiesis, restores BM erythropoiesis, and suppresses splenic erythropoiesis in LPS-treated mice.

(A) Serum concentrations of G-CSF in control mice, LPS-treated mice, and LPS-treated mice with anti-G-CSF treatment. (B) Representative images of femurs from control mice, LPS-treated mice, and LPS-treated mice with anti-G-CSF treatment (scale bar = 1 cm). (C) Quantification of total BM cells. (D) Representative flow cytometric plots of CD71⁺Ter119⁺ cells in the BMs of control mice, LPS-treated mice, and LPS-treated mice with anti-G-CSF treatment. (E) Flow cytometric quantification of CD71⁺Ter119⁺ erythroid cells in the BMs of control mice, LPS-treated mice, and LPS-treated mice with anti–G-CSF treatment. (F) Representative flow cytometric profiles of Ter119-APC and thiazole orange–stained cells from the BM of control mice, LPS-treated mice, and LPS-treated mice with anti–G-CSF treatment. (G) Flow cytometric quantification of reticulocytes (Ter119⁺ Thiazole orange⁺) in the BM of mice. (H) Flow cytometric quantification of erythroblasts (Ter119⁺ Thiazole orange⁺⁺) in the BMs of mice. (I) Representative flow cytometric plots of CD71⁺Ter119⁺ cells in the spleens of control mice, LPS-treated mice and LPS-treated mice with anti-G-CSF treatment. (J) Bar graphs showing quantification of CD71⁺Ter119⁺ cells in the spleens of control mice, LPS-treated mice, and LPS-treated mice with anti–G-CSF treatment. (K) Representative flow cytometric profiles of Ter119-APC and thiazole orange–stained cells from the spleen of control mice, LPS-treated mice, and LPS-treated mice with anti–G-CSF treatment. (L) Flow cytometric quantification of reticulocytes (Ter119⁺ Thiazole orange⁺) in the spleens from different treatment groups. (M) Flow cytometric quantification of erythroblasts (Ter119⁺ Thiazole orange⁺⁺) in the spleens from different treatment groups. (N) Representative flow cytometric plots of CD11b⁺Ly6G⁺ cells in the BMs of control mice, LPS-treated mice, and LPS-treated mice with anti–G-CSF treatment. (O) Flow cytometric quantification of CD11b⁺Ly6G⁺ neutrophils in the BMs of control mice, LPS-treated mice, and LPS-treated mice with anti–G-CSF treatment. (P) Peripheral neutrophil counts in control mice, LPS-treated mice, and LPS-treated mice with anti–G-CSF treatment. n = 3/group (D, E, F, G, H, I, J, K, L, M, N, O), n = 5/group (A, B, C, P). ***P < 0.001.

• Download figure
• Open in new tab
• Download powerpoint

Figure S7. Anti–G-CSF isotype control has no influence on BM and spleen that changed by LPS treatment.

(A) Representative images of femurs from WT control mice, LPS-treated mice and LPS-treated mice with isotype treatment (scale bar = 1 cm). (B) Quantification of total BM cells. (C) Representative flow cytometric plots of CD71⁺Ter119⁺ cells in the BMs of control, LPS-treated mice, and LPS-treated mice with isotype treatment. (D) Flow cytometric quantification of CD71⁺Ter119⁺ erythroid cells in the BMs of control, LPS-treated mice, and LPS-treated mice with isotype treatment. (E) Representative flow cytometric profiles of Ter119-APC and thiazole orange-stained cells from the BMs of control, LPS-treated mice, and LPS-treated mice with isotype treatment. (F) Flow cytometric quantification of reticulocytes (Ter119⁺ Thiazole orange⁺) in the BM of mice. (G) Flow cytometric quantification of erythroblasts (Ter119⁺ Thiazole orange⁺⁺) in the BMs of mice. (H) Representative flow cytometric plots of CD71⁺Ter119⁺ cells in the spleens of control, LPS-treated mice, and LPS-treated mice with isotype treatment. (I) Flow cytometric quantification of CD71⁺Ter119⁺ erythroid cells in the spleens of control, LPS-treated mice, and LPS-treated mice with isotype treatment. (J) Representative flow cytometric profiles of Ter119-APC and thiazole orange–stained cells from the spleens of control, LPS-treated mice, and LPS-treated mice with isotype treatment. (K) Flow cytometric quantification of reticulocytes (Ter119⁺ Thiazole orange⁺) in the spleens of mice. (L) Flow cytometric quantification of erythroblasts (Ter119⁺ Thiazole orange⁺⁺) in the spleens of mice. (M) Representative flow cytometric plots of CD11b⁺Ly6G⁺ cells in the BMs of control, LPS-treated mice, and LPS-treated mice with isotype treatment. (N) Flow cytometric quantification of CD11b⁺Ly6G⁺ cells in the BMs of mice. (O) Representative images of spleens from WT control mice, LPS-treated mice, and LPS-treated mice with isotype treatment. (P) Average weights of spleens from each group. (Q) Total cell numbers of spleens from each group. (R) Compressive strength of spleens from each group. n = 3/group (A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R). *P < 0.05, ***P < 0.001.

Knockout of TLR4 down-regulates G-CSF levels and restores the balance of erythropoiesis in LPS-treated mice

TLRs, which are expressed in immune effector cells and endothelial cells, trigger inflammatory responses by inducing production of inflammatory mediators. Endothelial cells can translate the signal of systemically spread pathogens into G-CSF release via TLR4 signaling after LPS challenge and are the prime sources of G-CSF that drives emergency bone marrow granulopoiesis (4, 35, 36). We speculated that TLR4 is the upstream factor that initiates the LPS-induced alteration of erythropoiesis. To test this hypothesis, we investigated erythropoiesis in the bone marrow and spleen during LPS treatment in TLR4-knockout mice. We observed that TLR4 deficiency almost prevented repression of bone marrow erythropoiesis (Fig 5A–G) and blocked splenic erythropoiesis in LPS-treated mice (Fig 5H–L). Next, we investigated the potential impact of TLR4 on G-CSF production and granulopoiesis of bone marrow in LPS-treated mice. In line with previous reports, TLR4^−/− mice showed low serum levels of G-CSF (Fig 5M), impaired emergency granulopoiesis of bone marrow (Fig 5N and O), and a dramatic decrease in blood neutrophil counts after LPS treatment (Fig 5P). Together, our findings suggest that TLR4-mediated G-CSF production plays an essential role in inflammation-associated splenic erythropoiesis.

• Download figure
• Open in new tab
• Download powerpoint

Figure 5. TLR4 knockout down-regulates G-CSF expression, abolishes the suppression of BM erythropoiesis, inhibits splenic erythropoiesis, and blocks emergency granulopoiesis in LPS-treated mice.

(A) Representative images of femurs from WT control mice, LPS-treated mice, and TLR4^−/− mice treated with LPS (scale bar = 1 cm). (B) Quantification of total BM cells. (C) Representative flow cytometric plots of CD71⁺Ter119⁺ cells in the BMs of WT control mice, LPS-treated mice, and TLR4^−/− mice treated with LPS. (D) Flow cytometric quantification of CD71⁺Ter119⁺ erythroid cells in the BMs of WT control mice, LPS-treated mice, and TLR4^−/− mice treated with LPS. (E) Representative flow cytometric profiles of Ter119-APC and thiazole orange–stained cells from the BM of WT control mice, LPS-treated mice, and TLR4^−/− mice treated with LPS. (F) Flow cytometric quantification of reticulocytes (Ter119⁺ Thiazole orange⁺) in the BM of mice. (G) Flow cytometric quantification of erythroblasts (Ter119⁺ Thiazole orange⁺⁺) in the BMs of mice. (H) Representative flow cytometric plots of CD71⁺Ter119⁺ cells in the spleens of WT control mice, LPS-treated mice, and TLR4^−/− mice treated with LPS. (I) Bar graphs showing quantification of CD71⁺Ter119⁺ cells in the spleens of WT control mice, LPS-treated mice and TLR4^−/− mice treated with LPS. (J) Representative flow cytometric profiles of Ter119-APC and thiazole orange-stained cells from the spleen of WT control mice, LPS-treated mice, and TLR4^−/− mice treated with LPS. (K) Flow cytometric quantification of reticulocytes (Ter119⁺ Thiazole orange⁺) in the spleens from different treatment groups. (L) Flow cytometric quantification of erythroblasts (Ter119⁺ Thiazole orange⁺⁺) in the spleens from WT control mice, LPS-treated mice, and TLR4^−/− mice treated with LPS. (M) Serum concentrations of G-CSF in WT control mice, LPS-treated mice, and TLR4^−/− mice treated with LPS. (N) Representative flow cytometric plots of CD11b⁺Ly6G⁺ cells in the BMs of WT control mice, LPS-treated mice, and TLR4^−/− mice treated with LPS. (O) Flow cytometric quantification of CD11b⁺Ly6G⁺ neutrophils in the BMs of WT control mice, LPS-treated mice, and TLR4^−/− mice treated with LPS. (P) Peripheral neutrophil counts in WT control mice, LPS-treated mice, and TLR4^−/− mice treated with LPS. n = 5/group (A, B, M, P), n = 3/group (C, D, E, F, G, H, I, J, K, L, N, O). ***P < 0.001.

G-CSF–induced splenic erythropoiesis relies on high levels of EPO

Erythropoiesis is driven by EPO that is mainly produced by renal EPO-producing cells in a hypoxia-inducible manner (29). To further study the mechanisms underlying G-CSF-induced erythropoiesis in the spleens of mice, we measured the circulating EPO levels. After G-CSF treatment (50 μg/kg), serum EPO levels in mice increased from 125 to 304 pg/ml (Fig 6A); in keeping with this result, we found that G-CSF treatment resulted in elevated EPO mRNA levels in the kidneys (Fig 6B). EPO is regulated by hypoxia-inducible factor (HIF); likewise, HIF-1α and HIf-2α were found to be up-regulated in the kidneys of mice treated with G-CSF (Fig 6C). Next, we assessed whether EPO is sufficient to induce splenic erythropoiesis. Wild-type mice were given four consecutive daily injections of EPO, after which a robust erythropoietic response was observed to take place in the spleen (Fig S8A–G). Single dose of EPO (200 U/kg) was administered to the mice; the results showed that hyperfunction of splenic erythropoiesis occurred in mice exposed to single dose of EPO (Fig S9).

• Download figure
• Open in new tab
• Download powerpoint

Figure 6. EPO mediates G-CSF–induced splenic erythropoiesis.

(A) Serum levels of EPO in control and G-CSF–treated mice. The mice were treated with G-CSF for 3 d. (B) Quantification of kidney EPO mRNA by quantitative reverse-transcribed polymerase chain reaction, normalized to β-actin mRNA. The mice were treated with G-CSF for 3 d. (C) Western blot analyses for the levels of HIF-1α and HIF-2α in kidneys. The mice were treated with G-CSF for 0, 1, 2, 3 d. Protein lysates were prepared from the kidney, and Western blot analyses were performed. (D) Protocol of anti-EPO blocking antibody treatment in G-CSF-treated mice. (E, F, G, H, I) Flow cytometric analysis of the splenic erythropoiesis in control mice, G-CSF–treated mice, and G-CSF–treated mice with anti-EPO treatment. (E) Representative flow cytometric plots of CD71⁺Ter119⁺ cells in the spleens. (F) Bar graphs showing quantification of CD71⁺Ter119⁺ cells in the spleens from different treatment groups. (G) Representative flow cytometric plots of Ter119-APC and thiazole orange-stained splenocytes from control mice, G-CSF–treated mice, and G-CSF–treated mice with anti-EPO treatment. (H) Flow cytometric quantification of reticulocytes (Ter119⁺ Thiazole orange⁺) in the spleens from different treatment groups. (I) Flow cytometric quantification of erythroblasts (Ter119⁺ Thiazole orange⁺⁺) in the spleens from different treatment groups. (J) Representative fields of burst-forming unit erythroid (BFU-E)–derived colonies in spleen cultures from control, G-CSF–treated mice, and G-CSF–treated mice with anti-EPO (×100 magnification; scale bar = 200 μm). (K) Quantification of the number of BFU-E-derived colonies in spleen cultures from different groups. (L) Protocol of red blood cell transfusions in G-CSF–treated mice. (M) Serum levels of EPO in WT control mice, G-CSF–treated mice, and G-CSF–treated mice with red blood cell transfusion. (N, O, P, Q, R) Flow cytometric analysis of the splenic erythropoiesis in control mice, G-CSF–treated mice, and G-CSF–treated mice with red blood cell transfusion. (N) Representative flow cytometric plots of CD71⁺Ter119⁺ cells in the spleens. (O) Bar graphs showing quantification of CD71⁺Ter119⁺ cells in the spleens from different treatment groups. (P) Representative flow cytometric profiles of splenocytes stained with anti-Ter119 and thiazole orange for each indicated group. (Q) Flow cytometric quantification of reticulocytes (Ter119⁺ Thiazole orange⁺) in the spleens from different treatment groups. (R) Flow cytometric quantification of erythroblasts (Ter119⁺ Thiazole orange⁺⁺) in the spleens from different treatment groups. (S) Representative fields of BFU-E–derived colonies in spleen cultures from control, G-CSF–treated mice, and G-CSF–treated mice with transfusion (×100 magnification; scale bar = 200 μm). (T) Quantification of the number of BFU-E–derived colonies in spleen cultures from different groups. n = 3/group (A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, S, T). **P < 0.01, ***P < 0.001.

• Download figure
• Open in new tab
• Download powerpoint

Figure S8. EPO injections significantly enhance splenic erythropoiesis and induce mild splenomegaly.

(A) Representative flow cytometric plots of CD71⁺Ter119⁺ cells in freshly isolated splenocytes from control and EPO-treated mice. (B) Bar graphs showing quantification of CD71⁺Ter119⁺ cells in the spleens of control and EPO-treated mice. (C) Representative flow cytometric profiles of Ter119-APC and thiazole orange–stained spleen cells from control and EPO-treated mice. (D) Flow cytometric quantification of reticulocytes (Ter119⁺ Thiazole orange⁺) in the spleens from different treatment groups. (E) Flow cytometric quantification of erythroblasts (Ter119⁺ Thiazole orange⁺⁺) in the spleens from different treatment groups. (F) Representative fields of burst-forming unit erythroid–derived colonies in spleen cultures from control and EPO-treated mice (×100 magnification; scale bar = 200 μm). (G) Quantification of the number of burst-forming unit erythroid–derived colonies in spleen cultures from control and EPO-treated mice. (H) Peripheral blood hematologic parameters of control and EPO-treated mice. Red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), and reticulocytes (Retics). (I) Representative photographs of spleens from control mice and EPO-treated mice. (J) Average weights of spleens from each group. (K) Total cell numbers of spleens from each group. (L) Compressive strength of spleens from each group. n = 5/group (A, B, C, D, E, I, J, K, L), n = 3/group (F, G), n = 7/group (H). *P < 0.05, **P < 0.01, ***P < 0.001.

• Download figure
• Open in new tab
• Download powerpoint

Figure S9. The effects of single dose of EPO (200 U/kg) on spleen.

(A) Representative flow cytometric plots of CD71⁺Ter119⁺ cells in the spleens of control and EPO-treated mice. (B) Bar graphs showing quantification of CD71⁺Ter119⁺ cells in the spleens of control and EPO-treated mice. (C) Representative flow cytometric plots of Ter119-APC and thiazole orange-stained splenocytes from control and EPO-treated mice. (D) Flow cytometric quantification of reticulocytes (Ter119⁺ Thiazole orange⁺) in the spleens from different treatment groups. (E) Flow cytometric quantification of erythroblasts (Ter119⁺ Thiazole orange⁺⁺) in the spleens from different treatment groups. n = 3/group (A, B, C, D, E). *P < 0.05.

Hematologic parameter analysis showed that serial injections of EPO increased red blood cell numbers, hemoglobin levels, hematocrits, and reticulocyte counts in mice (Fig S8H). To certify the cardinal role of EPO in splenic erythropoiesis, mice were treated with G-CSF together with anti-EPO neutralizing antibody (Fig 6D). Anti-EPO treatment reduced the increase in Ter119⁺CD71⁺ cells, reticulocytes, erythroblasts and the numbers of BFU-E in the spleens of mice treated with G-CSF (Figs 6E–K and S10A–E), indicating that neutralization of EPO with antibody inhibits G-CSF–induced splenic erythropoiesis.

• Download figure
• Open in new tab
• Download powerpoint

Figure S10. Anti-EPO isotype control has no influence on BM and spleen that changed by G-CSF treatment.

(A) Representative flow cytometric plots of CD71⁺Ter119⁺ cells in the spleens of control, G-CSF–treated mice, and G-CSF–treated mice with isotype treatment. (B) Flow cytometric quantification of CD71⁺Ter119⁺ erythroid cells in the spleens of control, G-CSF–treated mice, and G-CSF–treated mice with isotype treatment. (C) Representative flow cytometric profiles of Ter119-APC and thiazole orange–stained cells from the spleens of control, G-CSF–treated mice, and G-CSF–treated mice with isotype treatment. (D) Flow cytometric quantification of reticulocytes (Ter119⁺ Thiazole orange⁺) in the spleens of mice. (E) Flow cytometric quantification of erythroblasts (Ter119⁺ Thiazole orange⁺⁺) in the spleens of mice. (F) Representative images of spleens from WT control mice, G-CSF–treated mice, and G-CSF–treated mice with isotype treatment (scale bar = 1 cm). (G) Average weights of spleens from each group. (H) Total cell numbers of spleens from each group. (I) Compressive strength of spleens from each group. n = 3/group (A, B, C, D, E, F, G, H, I). ***P < 0.001.

In view of the above findings, it was of interest to establish that G-CSF suppressed bone marrow erythropoiesis, thus causing the anemia and renal hypoxia that stimulated secretion of renal EPO that subsequently promoted splenic erythropoiesis. Because red blood cell transfusions can correct anemia and tissue hypoxia (37, 38), we examined the impact of red blood cell transfusions on the response to G-CSF administration (Fig 6L). We transfused G-CSF–treated mice with red blood cells from WT mice and found that there was a significant reduction in serum EPO (Fig 6M) that was accompanied by a significant inhibition of splenic erythropoiesis (Fig 6N–T). We further examined the kinetics of peripheral red blood cell. Evaluation of hematologic parameters revealed that there were no differences in red blood cell numbers and hemoglobin levels between G-CSF-treated mice (on days 1, 2, 3, 4, 5, 6, and 9) and control mice (Fig S11). These results are somewhat contradictory. We speculate that EPO promotes splenic erythropoiesis and rapidly stimulates the release of splenic red blood cells. We further speculate that the oxygen carrying capacity of the red blood cell from splenic erythropoiesis is lower than bone morrow-derived red blood cell.

• Download figure
• Open in new tab
• Download powerpoint

Figure S11. The kinetics of red blood cell numbers and hemoglobin levels in G-CSF–treated mice.

(A) Peripheral red blood cell (RBC) counts in G-CSF–treated mice. (B) Peripheral hemoglobin (HGB) counts in G-CSF–treated mice. n = 6/group (A, B).

Adenine intake in mice induces progressive kidney damage (39). To acquire greater insight into the key role of the kidneys in G-CSF–induced splenic erythropoiesis, we used a model of adenine-induced chronic kidney injury. Treatment of mice with a 0.3% adenine diet induced continuous progressive kidney damage with increased serum urea and creatinine (Fig 7A and B). Renal histology revealed tubular atrophy, erosion of proximal tubular brush borders with flattening of the epithelium, and focal tubular epithelial hypertrophy (Fig 7C). As expected, adenine-induced chronic kidney injury resulted in lower serum EPO levels (Fig 7D) and inhibited splenic erythropoiesis in mice treated with G-CSF (Fig 7E–I). High-dose EPO injection rescued the erythropoietic function of spleens under kidney injury conditions in G-CSF–treated mice (Fig 7E–I). Taken together, these results indicate that renal EPO plays a key role in G-CSF–induced erythropoiesis.

• Download figure
• Open in new tab
• Download powerpoint

Figure 7. Adenine intake–induced renal dysfunction decreases serum EPO levels and inhibits splenic erythropoiesis in G-CSF–treated mice.

(A, B) Levels of blood urea nitrogen and serum creatinine in control mice, G-CSF–treated mice, and G-CSF–treated mice fed with adenine. (C) Representative histological photomicrographs of kidneys. Original magnification, top panels ×100, and bottom panels ×200. (D) Serum levels of EPO in control mice, G-CSF–treated mice, and G-CSF–treated mice fed with adenine. (E, F, G, H, I) Flow cytometric analysis of the splenic erythropoiesis in control mice, G-CSF–treated mice, G-CSF–treated mice fed with adenine and G-CSF–treated mice fed with adenine and with EPO treatment. (E) Representative flow cytometric plots of CD71⁺Ter119⁺ cells in the spleens. (F) Bar graphs showing quantification of CD71⁺Ter119⁺ cells in the spleens from different treatment groups. (G) Representative flow cytometric plots of Ter119-APC and thiazole orange–stained splenocytes from different treatment groups. (H) Flow cytometric quantification of reticulocytes (Ter119⁺ Thiazole orange⁺) in the spleens from different treatment groups. (I) Flow cytometric quantification of erythroblasts (Ter119⁺ Thiazole orange⁺⁺) in the spleens from different treatment groups. n = 3/group (A, B, C, D, E, F, G, H, I). **P < 0.01, ***P < 0.001, n.s., no significance.

G-CSF treatment results in dose-dependent splenomegaly that is associated with increased fragility of spleen in G-CSF–treated mice

Spleen is a fragile organ; severe splenomegaly may increase the risk for splenic rupture that is a potentially serious event (40, 41). To confirm the correlation between splenomegaly and splenic fragility in G-CSF–treated mice, we conducted a dose-finding experiment. We observed that G-CSF–treated mice developed progressively spleen enlargement with the incremental dosage of G-CSF administration (Fig 8A). Measurement of weight and cell number revealed significant increases in both splenic weights and splenocytes in mice treated with G-CSF from 5 to 100 μg/kg (Fig 8B and C); spleen/body weight ratios also increased in G-CSF–treated mice relative to control mice (Fig 8D). By contrast, there was no increase in the liver, lung, heart, kidney, or body weight in animals receiving G-CSF treatment (Fig S12). In an attempt to evaluate the fragility of spleen, we carried out a compression test on spleen (Fig 8E). The compressive strength (Fig 8F) from the splenic test indicated that G-CSF–treated mice underwent a progressive increase in splenic brittleness proportional to the increase in G-CSF dose. G-CSF–induced splenomegaly and increase of splenic brittleness were also time-dependent (Fig 8G–J). Single dose of G-CSF did not induced splenomegaly (Fig S2P–S). The spleen is surrounded by a capsule that contains collagen, elastic fibers, and smooth muscle and provides some rigidity to the spleen together with trabeculae that protrudes from capsule into splenic tissue (42, 43); rapid splenic enlargement can produce a thin and tense splenic capsule that is susceptible to rupture (44, 45). Therefore, we measured the thickness of splenic capsules by histology and found that the mean thickness of splenic capsules in G-CSF–treated mice (50 μg/kg) was 2.46 μm compared with 10.38 μm in control mice (Fig 8K and L). The thin capsule was accompanied by fewer and smaller trabeculae in G-CSF-treated mice (Fig 8M and N). The decreased thickness of splenic capsule and lack of splenic trabeculae may account for the increased fragility of spleens from G-CSF-treated mice.

• Download figure
• Open in new tab
• Download powerpoint

Figure 8. G-CSF treatment leads to splenomegaly and increases splenic fragility in mice.

(A) Macroscopic views of spleens in control and G-CSF–treated mice (scale bar = 1 cm). (B) Average weights of spleens in control and G-CSF–treated mice. (C) Total cell numbers of spleens in control and G-CSF–treated mice. (D) The ratios of spleen weight with respect to body weight. (E) Schema showing the compression test of spleen. (F) Compressive strength of spleens from each group. (G) Representative images of spleens from control and G-CSF-treated mice (scale bar = 1 cm). (H) Average weights of spleens from each group. (I) Total cell numbers of spleens from each group. (J) Compressive strength of spleens from each group. (K, M) Histopathological views of spleen tissues from the WT control mice and G-CSF–treated mice. Splenic sections were stained with H&E. The thickness of splenic capsule was measured under the microscope and the numbers of trabeculae were counted in histological photomicrographs. Original magnification, upper panels ×100 and lower panels ×200. (L) Quantification of thicknesses of splenic capsules. (N) Quantification of the numbers of trabecula in different groups (×100). n = 5/group (A, B, C, D, E, F, G, H, I, J, K, L, M, N). *P < 0.05, **P < 0.01, ***P < 0.001.

• Download figure
• Open in new tab
• Download powerpoint

Figure S12. G-CSF treatment has no influence on the liver, lung, heart, kidney, and body weight.

(A) Liver weights of control and G-CSF–treated mice. (B) Lung weights of control and G-CSF–treated mice. (C) Heart weights of control and G-CSF–treated mice. (D) Kidney weights of control and G-CSF–treated mice. (E) Body weights of control and G-CSF–treated mice. n = 5/group (A, B, C, D, E).

TLR4-mediated G-CSF production is essential for inflammation-associated splenomegaly in mice

Next, we tested to determine the pathological relevance of G-CSF in mice with splenomegaly during systemic inflammatory processes. Macroscopic analysis revealed that both E. coli-infected mice and LPS-treated mice developed spleen enlargement (Fig S13A and B). Comparison of weights and cell counts showed significant increases in splenic weight and splenocyte number of mice infected with E. coli or treated with LPS (Fig S13C–F). Both infection of mice with E. coli and treatment of mice with LPS characteristically decreased the compressive strength of the spleens (Fig S13G and H), indicating that mice bearing infection-related systemic inflammation undergo a progressive increase in splenic brittleness. The enhancements in spleen size, splenic weight, and splenocyte number were ameliorated by continuous neutralization of G-CSF in LPS-treated mice; importantly G-CSF neutralization decreased the splenic friability in LPS-treated mice (Figs 9A–D and S7O–R). Next, we investigated the potential impact of TLR4 on LPS-induced splenomegaly and observed that TLR4 deficiency almost prevented splenomegaly and increased splenic friability in LPS-treated mice (Fig 9E–H). Single treatment of LPS and single infection of E. coli were not associated with splenomegaly (Figs S5O–R and S6O–R). Together, our findings suggest that G-CSF mediates inflammation-induced splenomegaly.

• Download figure
• Open in new tab
• Download powerpoint

Figure S13. Either E. coli infection or LPS treatment induces splenomegaly and high fragility of spleen in mice.

(A) Representative images of spleens from control mice and E. coli-infected mice (scale bar = 1 cm). (B) Representative images of spleens from control mice and LPS-treated mice (scale bar = 1 cm). (C) Average weights of spleens from control mice and E. coli–infected mice. (D) Average weights of spleens from control mice and LPS-treated mice. (E) Total cell numbers of spleens from control mice and E. coli–infected mice. (F) Total cell numbers of spleens from control mice and LPS-treated mice. (G) Compressive strength of spleens from control mice and E. coli–infected mice. (H) Compressive strength of spleens from control mice and LPS-treated mice. n = 5/group (A, B, C, D, E, F, G, H). ***P < 0.001.

• Download figure
• Open in new tab
• Download powerpoint

Figure 9. TLR4-mediated G-CSF production contributes to splenomegaly and high fragility of spleen during systemic inflammation in mice.

(A) Representative images of spleens from WT control mice, LPS-treated mice, and LPS-treated mice with anti–G-CSF treatment (scale bar = 1 cm). (B) Average weights of spleens from each group. (C) Total cell numbers of spleens from each group. (D) Compressive strength of spleens from each group. (E) Representative images of spleens from WT control mice, LPS-treated mice, and TLR4^−/− mice treated with LPS (scale bar = 1 cm). (F) Average weights of spleens from each group. (G) Total cell numbers of spleens from each group. (H) Compressive strength of spleens from each group. n = 5/group (A, B, C, D, E, F, G, H). ***P < 0.001.

Splenic erythropoiesis partially contributes to G-CSF–induced splenomegaly and high splenic fragility in mice

Splenomegaly is a complication of splenic erythropoiesis (34, 46). Next, we sought to address potential roles of splenic erythropoiesis in G-CSF–induced splenomegaly and high fragility of spleen. We first treated mice with G-CSF together with anti-EPO neutralizing antibody, and we found that neutralization of EPO only slightly inhibited the progression of splenic enlargement (Figs 10A–C and S10F–H) and mildly increased the compressive strength of spleens (Figs 10D and S10I). These results indicate that EPO neutralization alleviates but does not reverse splenomegaly and high fragility of spleen induced by G-CSF. Similarly, red blood cell transfusions slightly mitigated G-CSF-induced splenomegaly and fragility rupture of spleen (Fig 10E–H). To further validate the role of EPO in splenomegaly and splenic fragility, we treated WT mice with EPO and found that the splenic weight, splenocyte number, and splenic fragility mildly increased after daily injection of EPO (200 U) for four consecutive days (Fig S8I–L). We then investigated the effects of kidney injury on G-CSF-induced splenic enlargement and fragility rupture. In contrast to EPO neutralization and red blood cell transfusion, adenine-induced kidney injury almost blocked G-CSF–associated splenomegaly and significantly increased the compressive strength of spleens in mice treated with G-CSF (Fig 10I–L). To unequivocally determine the role of splenic erythropoiesis in G-CSF–induced splenomegaly and fragility rupture of spleen, we monitored the effects of high-dose EPO injections on the spleens in G-CSF–treated mice with kidney injury. The spleen weight, splenocyte number, and splenic fragility in mice with kidney injury treated with G-CSF moderately increased after high-dose EPO administration (Fig 10I–L). These results indicate that splenic erythropoiesis partially accounts for G-CSF–induced splenomegaly and splenic fragility.

• Download figure
• Open in new tab
• Download powerpoint

Figure 10. EPO neutralization, red blood cell transfusion, and adenine-induced kidney injury alleviate splenomegaly in G-CSF–treated mice.

(A) Representative images of spleens from WT control mice, G-CSF–treated mice, and G-CSF–treated mice with anti-EPO treatment (scale bar = 1 cm). (B) Average weights of spleens from each group. (C) Total cell numbers of spleens from each group. (D) Compressive strength of spleens from each group. (E) Representative images of spleens from WT control mice, G-CSF–treated mice, and G-CSF–treated mice with red blood cell transfusion (scale bar = 1 cm). (F) Average weights of spleens from each group. (G) Total cell numbers of spleens from each group. (H) Compressive strength of spleens from each group. (I) Representative images of spleens from control mice, G-CSF–treated mice, G-CSF–treated mice fed with adenine, and G-CSF–treated mice fed with adenine and with EPO treatment (scale bar = 1 cm). (J) Average weights of spleens from each group. (K) Total cell numbers of spleens from each group. (L) Compressive strength of spleens from each group. n = 5/group (A, B, C, D, E, F, G, H, I, J, K, L). *P < 0.05, **P < 0.01, ***P < 0.001.