Article Figures & Data
Figures
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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).
- 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.
- 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.
- 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).
- 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.
- 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.
- 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.
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