| IntroductionGranulocyte colony-stimulating factor(G-CSF)is a glycoprotein that is secreted by endothelial cells,macrophages,and fibroblasts.G-CSF binds to the specific receptors(G-CSFR)on the surface of granulocytic progenitors and mature neutrophils,activates the downstream signaling pathways,promotes the proliferation and differentiation of bone marrow granulocytic precursors into neutrophils,and enhances the chemotaxis,phagocytosis and killing functions of mature neutrophils.Recombinant human G-CSF is widely used in patients with congenital and acquired neutropenia to prevent and treat infection.Moreover,as the main agent to mobilize hematopoietic stem and progenitor cell(HSPC)for transplantation,G-CSF mobilizes HSPC from the bone marrow into the circulation.Neutrophils play an important role in the resistance to infection of pathogenic microorganism.When the body is infected by bacteria,bacteria can stimulate the endothelial cells to secret G-CSF,which drives emergency granulopoiesis,mobilizes neutrophils to release from the bone marrow into the peripheral blood,and causes the increase of the number of neutrophils in the peripheral blood.Under the action of chemokines produced in the inflammatory region,peripheral blood neutrophils infiltrate into the infected site,phagocytosis and kill bacteria.Erythropoiesis is a carefully orchestrated process that culminates in the generation of new erythrocytes to replace the old red blood cells at a constant rate.Under steady-state conditions,bone marrow is the primary site of adult erythropoiesis Infection-related inflammation suppresses bone marrow erythropoiesis and induces anemia.The occurrence of anemia during infection often correlates with poor prognosis.Anemia of inflammation caused by infection reduces the quality of life of patients and increases the medical burden of infected patients.The mechanism of inflammation-induced suppression of bone marrow erythropoiesis has not been fully elucidated.Interferon-y(IFN-γ),tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6)may participate in the suppression of bone marrow erythropoiesis in the process of inflammation.G-CSF can inhibit bone marrow erythropoiesis.However,it has not been systematically reported whether the opposite changes between neutrophils and erythrocytes are intrinsically related in the process of systemic inflammation caused by bacterial infection,and whether G-CSF plays an important role in this process.Therefore,during systemic inflammation caused by infection,it is of great significance to explore the role of G-CSF in the shift of bone marrow hematopoietic function.Clarifying the effect of G-CSF on the disorder of bone marrow erythropoiesis in the process of systemic inflammation is of critical importance in patients with infection and provides a new target and theoretical basis for the clinical treatment of anemia of inflammation.ObjectiveTo investigate the role of G-CSF on bone marrow erythropoiesis in infection-related systemic inflammation,and to explore the possible mechanism by which G-CSF suppresses bone marrow erythropoiesis.Methods1.C57BL/6 mice were intravenously injected with 1×106 E.coli once every 2 days for 9 days.The mice were injected on days 0,2,4,6,and 8 respectively.On day 9,blood was obtained by cardiac puncture.Routine blood tests were performed on EDTA-anticoagulated blood.Serum was harvested,and G-CSF concentrations were determined by cytometric bead array(mouse G-CSF flex set).After mice were euthanized,the femurs were dissected and the bone marrow cells were collected.2.C57BL/6 mice were intraperitoneally injected with 0.5mg/kg LPS twice daily for 9 days.On day 9,blood was obtained by cardiac puncture.Routine blood tests were performed on EDTA-anticoagulated blood.Serum was harvested,and G-CSF concentrations were determined in the different groups.After mice were euthanized,the femurs were dissected and the bone marrow cells were collected.3.C57BL/6 mice were subcutaneously injected with G-CSF(1,2,5,10,20,50,or 100 μ/kg)twice daily for 9 days.After mice were euthanized,the femurs were dissected and the bone marrow cells were collected.4.C57BL/6 mice were subcutaneously injected with G-CSF(50 μg/kg).The mice were injected for 0,6,12,24,48,72,144 and 216 h respectively.The mice were injected twice daily from 24 hours to 216 hours for 9 days.After mice were euthanized,the femurs were dissected and the bone marrow cells were collected.5.C57BL/6 mice were subcutaneously injected twice daily with G-CSF(50 μg/kg)for nine days,and then G-CSF was stopped for 3,6,9,12,and 15 d respectively After mice were euthanized,the femurs were dissected and the bone marrow cells were collected6.C57BL/6 mice and TLR4-/-mice were intraperitoneally injected with 0.5mg/kg LPS twice daily for 9 days.Blood was collected by cardiac puncture,and the serum was collected by centrifugation.After mice were euthanized,the femurs were dissected and the bone marrow cells were collected.7.C57BL/6 mice were intraperitoneally injected with 0.5mg/kg LPS twice daily for 9 days,and the G-CSF neutralizing antibody(2.5 mg/kg)were intraperitoneally injected on days 2,5,and 8 during LPS administration.Blood was collected by cardiac puncture,and the serum was collected by centrifugation.After mice were euthanized,the femurs were dissected and the bone marrow cells were collected.8.The bone marrow cells were labeled with APC Rat Anti-mouse Ter119 and PE Rat Anti-mouse CD71,and the proportion of CD71+Ter119+cells was analyzed by flow cytometry.9.The bone marrow cells were labeled with APC Rat Anti-Mouse Ter119 and Thiazole orange,and the proportions of reticulocytes(Ter119+Thiazole orange+)and erythroblasts(Ter119+Thiazole orange++)were analyzed by flow cytometry.10.The bone marrow cells were labeled with APC Rat Anti-Mouse CD11b and PE Rat Anti-Mouse Ly6G,and the proportion of CD11b+Ly6G+cells was analyzed by flow cytometry.11.BFU-E(burst forming unit-erythroid)and CFU-GM(colony forming unit-granulocyte and macrophage)of mice bone marrow cells were seeded into methylcellulose-based media.Colony formation was observed under microscope.12.Bone marrow cells from control and G-CSF-treated mice were stained with specific antibodies for GATA-1,p-JAK2 and p-STAT3,and the expression of GATA-1 and the phosphorylation levels of JAK2 and STAT3 were detected by flow cytometry.Results1.After E.coli was injected into the tail vein to induce systemic infection,the femurs of mice became pale color and the number of cells in the bone marrow decreased.The proportion of CD71+Ter119+cells,reticulocytes and erythroblasts in bone marrow cells decreased,and the number of peripheral blood erythrocytes decreased in E.coli-infected mice.E.coli infection increased serum levels of G-CSF in mice.The proportion of CD11b+Ly6G+cells in bone marrow increased,and the number of neutrophils in peripheral blood increased in E.coli-infected mice2.During lipopolysaccharide(LPS)-induced systemic inflammation in mice,the femurs exhibited pale color,and the cell number of bone marrow decreased.The proportion of CD71+Ter119+ cells,reticulocytes,and erythroblasts in bone marrow decreased,and the peripheral blood erythrocytes decreased in LPS-treated mice.LPS treatment increased serum levels of G-CSF in mice.The proportion of CD11b+Ly6G+cells in bone marrow increased,and the number of neutrophils in peripheral blood increased in LPS-treated mice3.The mouse bones became progressively paler in response to the gradually increasing doses of G-CSF(0,1,2,5,10,20,50 and 100μg/kg),and the number of bone marrow cells decreased successively.The percentage of CD71+Ter119+cells,reticulocytes,and erythroblasts in the bone marrow of mice gradually decreased with increasing doses of G-CSF.The proportion of CD11b+Ly6G+cells in bone marrow increased gradually,and the number of neutrophils in peripheral blood increased gradually in G-CSF-treated mice4.The femur of mice treated with G-CSF(50μg/kg)gradually showed pale color and the total number of bone marrow cells decreased with the extension of injection time(0,6,12,24,48,72,144 and 216h).The proportion of CD71+Ter119+cells,reticulocytes,and erythroblasts decreased with a time-dependent manner in G-CSF-treated mice5.The CD71+Ter119+cells,reticulocytes,and erythroblasts in the bone marrow were restored after G-CSF withdrawal,and the bone color and bone marrow cell number also recovered with the extension of withdrawal time in mice.6.After intraperitoneal injection of LPS in TLR4 knockout mice,the serum concentration of G-CSF in TLR4-/-mice did not increase significantly,and the color of bone and cell number of bone marrow did not change in TLR4-/-mice After LPS treatment,the proportion of CD71+Ter119+cells in TLR4-/-mice did not significantly decrease,and the proportions of reticulocytes and erythroblasts did not significantly decrease.After LPS stimulation,the CD11b+Ly6G+cells in bone marrow of TLR4-/-mice was not significantly increased,and the number of neutrophils in peripheral blood was not significantly increased.7.In the mouse model of systemic inflammation induced by LPS,G-CSF neutralization reduced serum G-CSF concentrations,restored the color of bone and bone marrow cell numbers.The proportion of CD71+Ter119,cells,reticulocytes,erythroblasts,CD11b+Ly6G+cells in bone marrow,and neutrophils in peripheral blood were all maintained at normal levels after anti-G-CSF treatment in LPS-treated mice8.In mice treated with G-CSF(50μg/kg)for 9 days,bone marrow cells from G-CSF-treated mice formed lower numbers of BFU-E colonies than the controls,and the numbers of CFU-GM derived from the bone marrow of G-CSF-treated mice were increased.9.In mice treated with G-CSF(50μg/kg)for 2 days,the expression of GATA-1 was decreased and the phosphorylations of JAK2 and STAT3 were increased in bone marrow cells.ConclusionDuring bacterial infection-induced systemic inflammation,LPS increases the levels of G-CSF in mice by activating the signaling pathway of TLR4.Elevated levels of G-CSF promote granulopoiesis by up-regulating the phosphorylation of JAK2 and STAT3,and inhibit bone marrow erythropoiesis by down-regulating the expression of GATA-1 in bone marrow cells.Blockade of G-CSF function can alleviate suppression of erythropoiesis in bone marrow caused by systemic inflammation. |
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