Mechanisms Of Kupffer Cells And Liver Sinusoidal Endothelial Cells Promoting Liver Hematopoiesis

Purposes:It has been established that the liver is the major hematopoietic organ during fetal period.After birth,hematopoietic stem cells reside primarily in the bone marrow.In adults,extramedullary hematopoiesis occurs in the liver and other solid organs when hematopoiesis in the bone marrow fails,as a result of some pathological conditions,suggesting that adult liver contains hematopoietic stem and progenitor cells,which are associated with long-term hematopoietic reconstitution activity.However,the characteristics of adult liver hematopoiesis,the composition of liver hematopoietic microenvironment and the regulatory mechanism of liver microenvironment on liver hematopoiesis remain unknown.In the present study,We detected the presence of hematopoietic stem and progenitor cells in the adult liver:lin-sca-l+c-kit+(LSK,contains hematopoietic stem cells and multipotent progenitors)cells and lin-mac-1+sca-1+(LMS)cells,which are specific in liver tissues different from bone marrow.HSPC transplantation experiments were used to confirm that adult murine liver LSK and LMS cells differentiate into both myeloid cells and lymphocytes(preferentially T cells)compared with bone marrow HSPCs.We further explored how liver microenvironment promotes liver hematopoiesis and T lymphocyte differentiation,and which factors are involved in this process.On the one hand,we found that kupffer cells,liver resident macrophages,could promote the differentiation of adult liver LSK cells into lymphocytes and myeloid cells,mainly T lymphocytes and B lymphocytes.A blockade of intercellular cell adhesion molecule-1(ICAM-1)in a liver HSPC and kupffer cell coculture system impaired the adhesion,expansion,and differentiation of HSPCs..These results suggest that kupffer cells play an important role in maintaining and promoting liver hematopoiesis.On the other hand,we further confirmed that adult hepatic hematopoietic stem cells have a group of hematopoietic cells-LMS cells,which are different from bone marrow.And liver sinusoidal endothelial cells,as an important component of liver hematopoietic microenvironment could promote the differentiation of liver LMS cells via notch signal,and T lymphocyte differentiation is dominant.In Cconclusion,these results suggest that the cellular and molecular in the liver microenvironment play an important role in maintaining and promoting adult liver hematopoiesis.These findings provide important insight into understanding liver extramedullary hematopoiesis and its significance.It provides a theoretical basis for the treatment of some liver diseases,such as hepatitis,non-alcoholic fatty liver disease(NAFLD)and hepatocellular carcinoma(HCC).Methods:1.To detect and compare the proportion of hematopoietic stem progenitor cells(LSK and LMS)in different organs by flow cytometry.2.To analysis the colony formation ability of the liver mononuclear cells and LSK cells using a methylcellulose semi-solid medium assay.3.To confirm the hematopoietic reconstitution ability of adult liver hematopoietic stem progenitor cells.A total of 2 x 104 LSK cell suspensions obtained from either the liver or bone marrow of donor CD45.2+mice by flow cytometry were mixed with 2 x 105 bone marrow mononuclear cell suspensions from CD45.1 mice and injected into lethally irradiated CD45.1+ recipients to detect the proportion of CD45.2+cells,lymphocytes and myeloid cells in peripheral blood of CD45.1+ recipients at different time points by flow cytometry.4.Using a LPS-stimulated murine extramedullary hematopoiesis model to promote adult liver hematopoiesis.5.To observe the effect of clearance of kupffer cells on liver extramedullary hematopoiesis.Intravenously injected CL into LPS-treated mice to delete macrophages and kupffer cells.6.To assess the level of hematopoietic growth factor and pro-inflammatory factors secreted by kupffer cells by ELISA.7.To detect the differentiation of liver hematopoietic stem progenitor cells into lymphocytes and myeloid cells by co-culture and flow cytometry at different time points.8.To detect the expression of ICAM-1 and VCAM-1 on kupffer cells and the corresponding ligands LFA-1 and VLA-4 on LSK cells by flow cytometry.9.The liver LSK cells were sorted and cocultured with kupffer cells in the presence of SCF with or without an anti-ICAM-1 antibody for 14 days,and then the differentiated lymphoid and myeloid cells were detected by flow cytometry.10.The expression of notch ligand in liver sinusoidal endothelial cells and hematopoietic regulatory factor in liver microenvironment was detected by real-time fluorescence quantitative PCR.11.To detect the expression of notch receptors notchl and notch2 on LMS cells by flow cytometry.12.To detect the expression of notch activated form NICD on LMS cells in co-culture system by flow cytometry.13.To detect the effect of notch signal inhibitor(LY411575)on the differentiation of LMS into T lymphocyte by flow cytometry.Results:1 Mechanisms of kupffer cells promoting liver hematopoiesis1.1 The adult murine liver contains hematopoietic progenitor cellsTo detect and compare the proportion of LSK cells in liver and bone marrow of mice at different developmental stages by flow cytometry.A small number of LSK cells were observed in adult liver.Using methylcellulose semi-solid culture experiment,we found that the LSK cells derived from the liver had the ability to form a certain number of GM-CFU and M-CFU clones.However,the total numbers of GM-CFU and M-CFU clones derived from the liver LSK cells were significantly lower than those isolated from the bone marrow.These results suggest that there are hematopoietic stem progenitor cells in adult liver.1.2 Murine adult liver LSK cells are capable of generating both lymphoid and myeloid cellsWe observe that liver-derived LSK cells could reconstruction lymphocytes and myeloid cells in lethally irradiated mice by hematopoietic transplantation experiments,Although the hematopoietic reconstitution ability of liver-derived LSK cells was significantly weaker than that of bone marrow.Compared with bone marrow,liver-derived LSK cells preferentially differentiate into T lymphocytes.Bone marrow-derived LSK cells mainly produce B lymphocytes and produce less T lymphocytes.We also found that liver-derived LSK cells mainly differentiated into lymphocytes and myeloid cells in the liver,but rarely migrated to bone marrow.However,bone marrow-derived LSK cells can migrate to both bone marrow and liver.1.3 Kupffer cells promote LPS-induced liver hematopoiesisExtramedullary hematopoietic model was induced by intraperitoneal injection of LPS for 3 days.The number of LSK cells and long-term hematopoietic stem cells(LT-HSC)in liver was detected by flow cytometry.The absolute number of LPS treated mice liver LSK and LT-HSC cells increased significantly compared with the control group.It suggests that LPS can induce liver extramedullary hematopoiesis.After deleting kupffer cells with chlorophosphate liposomes,the number of liver LSK cells and long-term hematopoietic stem cells(LT-HSC)induced by LPS decreased significantly,when the deleting kupffer cells group compared with the control group.This suggests that kupffer cells play an important role in LPS-induced liver extramedullary hematopoiesis.1.4 Kupffer cells sustain liver HSPCs to differentiate into T and B cellsThrough the co-culture experiment of kupffer cells and LSK cells,we observed that when kupffer cells were present in the co-culture system,the liver LSK cells could differentiate into lymphocyte and myeloid cells.While the liver LSK cells could not differentiate into lymphocyte and myeloid cells only in the presence of culture medium and SCF.If LPS was added to the co-culture system,promoting-differentiation effect of kupffer cells could be enhanced.This suggests that kupffer cells can promote the differentiation of liver hematopoietic stem progenitor cells.1.5 Kupffer cells promote the proliferation of liver HSPCsUsing LPS-induced liver extramedullary hematopoietic model,we observed that LPS treatment increased the proportion of liver Ki-67+LSK cells compared with the control group.After clearing kupffer cells,the proportion of Ki-67+LSK cells significantly decreased in LPS treatment group.These results suggest that kupffer cells can promote the proliferation of liver hematopoietic stem progenitor cells.1.6 Kupffer cells promote LPS-induced liver hematopoiesis and lymphocyte differentiation via ICAM-1 and LFA-1 interactionThe expression of adhesion molecules on kupffer cells was detected by flow cytometry using LPS-induced liver extramedullary hematopoietic model.We found that the expression of ICAM-1 increased significantly when LPS treatment group compared with the control group,but the expression of VCAM-1 did not change.We further examined the expression of LFA-1 and VLA-4,which are the corresponding ligands for ICAM-1 and VCAM-1,respectively,on LSK cells.The expression of LFA-1,but not VLA-1,was substantially elevated following LPS treatment We found that the total number of LSK cells,Lin-cells,CD3+ T cells and CD19+B cells significantly declined following the ICAM-1 blockade compared with the control group.This suggests that kupffer cells can promote LPS-induced liver hematopoiesis and lymphocyte differentiation via ICAM-1/LFA-1 interaction.2 Mechanisms of liver sinusoidal endothelial cells promoting liverhematopoiesis2.1 There are a group of LMS cells in the adult liver which are different from those in bone marrowFlow cytometry was used to detect the proportion of LMS cells in the fetal liver,adult liver,spleen and bone marrow.The hematopoietic reconstitution ability of fetal liver LMS cells was verified by hematopoietic transplantation experiment.We observed that the fetal liver contained LMS cells which exhibit hematopoietic function.We further found that there were still a high proportion of LMS cells in adult liver.However,LMS cells were hardly detected in the spleen and bone marrow.It is suggested that LMS cells may only exist in fetal and adult liver,and adult liver LMS cells may originate from fetal liver rather than bone marrow.2.2 The adult liver LMS cells are derived from fetal liverFetal liver and bone marrow mononuclear cells were transplanted to lethally irradiated mice.The generation of donor-derived(CD45.1+)LMS cells in the liver of CD45.2+recipient mice was detected at the 4th week after fetal liver mononuclear cells transplantation.The results showed that fetal liver-derived mononuclear cells could reconstruction LMS cells at the 4th week after transplantation,but bone marrow-derived mononuclear cells could not reconstruction LMS cells.We further examined the reconstitution of LMS cells in various organs of recipient mice after fetal liver mononuclear cells transplantation.We found that LMS cells could only reconstitute in the liver,but can hardly be detected in bone marrow,spleen and peripheral blood after fetal liver mononuclear cells transplantation.This suggests that adult liver LMS cells are derived from fetal liver rather than bone marrow.2.3 Adult mouse liver LMS cells could generate both lymphoid and myeloid cellsBy adult liver LMS cells transplantation experiments,we found that donor-derived CD45.1+ LMS cells could be detected in peripheral blood of recipient mice at the 4th week after transplantation and lasted until 7 weeks.We further observed adult mouse liver LMS cells could generate both lymphoid and myeloid cells.The results showed that donor-derived CD45.1+ cells were detected in the liver,spleen and bone marrow of recipient mice,and that donor-derived cells could differentiate into lymphoid cells(CD3+T,CD19+B,NK1.1+ T cells)and CD11b+myeloid cells in recipient liver,but mainly CD3+ T cells.This suggests that adult liver LMS cells can differentiate into lymphocytes and myeloid cells.2.4 Liver sinusoidal endothelial cells promote LMS cells to differentiate into lymphocytes and myeloid cellsBy co-culturing of liver sinusoidal endothelial cells with LMS cells,liver sinusoidal endothelial cells can promote the differentiation of LMS cells into CD3+ T,CD19+B,NK1.1+ NK lymphocytes cells and CDllb+ myeloid cells in vitro,and mainly produce CD3+ T cells.These results suggest that liver sinusoidal endothelial cells can promote the differentiation of LMS cells.2.5 Liver sinusoidal endothelial cells promote the differentiation of LMS cells into T cells via notch signaling.We further compared the expression of notch ligand Jag1,Jag2,DLL 1,DLL3 and DLL4 genes in liver sinusoidal endothelial cells in the single culture group(LSEC)and the co-culture group(LSEC+LMS).The results showed that the expression of DLL1 in LSEC+ LMS group was higher than that in LSEC group,there was no difference in the expression of Jagl and DLL4,the expression of Jag2 decreased,and the expression of DLL3 was not detected.This suggests that liver sinusoidal endothelial cells may promote the differentiation of LMS cells into CD3+ T cells via notch signaling.Next,we further examined the expression of notch receptors notch1 and notch2 in LMS cells.We found that LMS cells expressed a certain proportion of notch1 and notch2.We then examined the proportion of differentiated lymphocytes in co-culture of liver sinusoidal endothelial cells and LMS cells.We observed that the percentage of CD3' T cells decreased in co-culture system and the percentage of CD19+ B cells increased when notch signal inhibitor LY411575 treatment group compared with control group.In conclusion,these phenomena suggest that liver sinusoidal endothelial cells promote the differentiation of LMS cells into T cells through via notch signaling.Conclusion and Significance:1.Adult liver contains LSK cells and liver-specific LMS cells different from bone marrow.2.Liver LSK cells and LMS cells have the ability to differentiate into lymphocyte and myeloid cells,and T lymphocyte differentiation is dominant.3.This study is the first to find that kupffer cells can promote the proliferation of LSK cells in LPS-induced liver extramedullary hematopoietic model.In addition,kupffer cells can promote the differentiation of LSK cells into lymphocytes and myeloid cells,mainly T and B lymphocytes.LPS can enhance promoting differentiation effect of kupffer cells.Kupffer cells promote the retention,proliferation and differentiation of LSK cells in the liver via the interaction of ICAM-1/LFA-1.4.Liver sinusoidal endothelial cells can promote the differentiation of LMS cells into lymphocytes and myeloid cells and T lymphocyte differentiation is dominant.Liver sinusoidal endothelial cells promote the differentiation of LMS cells into T lymphocytes via notch signal.