Effect On NK Cells Induced By Myeloid-derived Suppressor Cells In Acute Fulminate Hepatitis Of Mice

Acute fulminate hepatitis has characteristic of rapid onset, severe illness and high mortality. The abnormal immune response is its typical feature. Changes in immune system function in the occurrence of hepatitis, development and prognosis are closely linked. The liver contains large amounts of cells involved in innate immune responses, such as NK cells, MΦ, NKT and ydT cells. In recent years, researchers pay more and more attention on inhibition of immune cell subsets with the phenotype of Gr1+CD11b+ myeloid derived suppressor cells (MDSCs). MDSCs are a heterogeneous population of cells that consists of myeloid progenitor cells and immature myeloid cells (IMCs). MDSCs expand during cancer, inflammation and infection and have function of immunoregulation. This project is to study the effects on myeloid derived suppressor cells to NK cells in innate immunity with the development of acute fulminate hepatitis.Combining the existing research reports, we use C57/BL6 mice as experimental animals and built a model of acute fulminate hepatitis caused by toxins. Based on this model, we measured the proportion MDSCs and of NK cells and the activity of NK cells during the development of hepatitis, to study the effect on MDSCs to NK cells and their activity during the development of hepatitis, to get more understanding on pathogenesis of acute fulminate hepatitis and treatment for hepatitis and to provide new ideas.Part I Preparation and identification of acute fulminate hepatitis mice modelUsing C57BL/6 mice as experimental animal and i.p. co-inject the mixture of lipopolysaccharide (LPS) (5μg/kg) and D-galactosamine (D-GalN) (450mg/kg) to prepara acute fulminate hepatitis mice model. This model is induced by endotoxin poisoning assisted by D-GalN to damage the liver. We detected the occurrence with the development of acute fulminate hepatitis mice model.First, we successfully prepared acute fulminate hepatitis mice model and observed survival time of the acute fulminate hepatitis mice.25% of the modeling mice died after 6.5h,75% of the modeling mice died after 7h-8h and all died within 10h. Using this phenomenon, we further examined serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels of the mice after the model 0h,1.5h,3h and 6h. Data showed that serum AST and ALT levels were significantly increased along with the time of co-injected with LPS/D-GalN, and serum AST and ALT 6h levels were significantly higher than the serum AST and ALT 1.5h and 3h levels. We also did pathology analyze of the mice liver at different time points. Liver biopsy co-injected with LPS/D-GalN 1.5h showed liver cells had many nuclear fission, there was scattered neutrophil infiltration. Liver biopsy co-injected with LPS/D-GalN 3h showed liver cells had more nuclear fission, with scattered infiltration of neutrophils, some areas were infiltrated with small focal of inflammatory cells and some liver cells were died. Liver biopsy co-injected with LPS/D-GalN 6h showed liver cells had more nuclear fission, there were more neutrophil infiltration, some areas were infiltrated with small focal of inflammatory cells and surrounded by a large number of dead liver cells. Results showed liver inflammation significantly increased with the development of acute fulminate hepatitis model.Part II The effect on MDSCs to NK cells and their activity with the development of acute fulminate hepatitis We isolated mononuclear cells from liver, spleen, bone marrow or lymph node tissue in the acute fulminate hepatitis model 0h,1.5h,3h and 6h. The cells were stained with FITC-anti-Grl and PE-anti-CD11b antibody and we measured the proportion of Gr1+CD11b+MDSCs cells through flow cytometry. We use PE-anti-CD3e and PE-Cy5-anti-NK1.1 antibody to stain the liver mononuclear cells and measured the proportion of CD3-NK1.1+NK cells at different time points. We use FITC-anti-NKG2D, PE-anti-CD3e and PE-Cy5-anti-NK1.1 antibody to stain the mononuclear cells isolated from the liver, spleen and bone marrow and measured the proportion ofNKG2D+NK cells and mean fluorescence intensity (MFI) of NKG2D at different time points.Results showed that MDSCs had significant accumulation in the liver, spleen, bone marrow and lymph node tissue with the development of acute fulminate hepatitis. At the same time, the number of liver NK cells and activated NK cell receptor NKG2D increased significantly with the development of acute fulminate hepatitis, the MFI of NK cells activation receptor NKG2D enhanced significantly with the development of acute fulminate hepatitis. This demonstrated that MDSCs and NK cells were gradually increased and the activity of NK cells strengthened with the development of fulminate hepatitis.PartⅢThe effect on MDSCs to NK cells after depletion of MDSCs with the development of acute fulminate hepatitisUsing Rb6 hybridoma to prepare monoclonal antibodies with the function of depletes MDSCs and then identified anti-Gr-1 mAb in vivo. After C57BL/6 mice injected with the monoclonal antibody, the proportion of Gr1+CD11b+MDSCs cells were significantly decreased to 1% in liver, spleen and bone marrow tissues.Using anti-Gr-1 mAb to prepare MDSCs depletion mice, on this basis, to use PE-anti-CD3e and PE-Cy5-anti-NKl.1 antibody to stain mononuclear cells isolated from the liver to measure the proportion of CD3-NK1.1+NK cells with the acute fulminate hepatitis model 6h. And we used FITC-anti-NKG2D, PE-anti-CD3e and PE-Cy5-anti-NK1.1 antibody to stain mononuclear cells isolated from the liver, spleen and bone marrow to measure the proportion of NKG2D+NK cells and MFI of NKG2D with the acute fulminate hepatitis model 6h.Results showed that the proportion of NK cells in mice liver tissue after depleted MDSCs with the acute fulminate hepatitis model 6h significantly higher than that of normal mice with the acute fulminate hepatitis model 6h. In addition, after removing the MDSCs in vivo, the proportion of activation of NK cell receptor NKG2D and MFI of NKG2D were significantly reduced with the acute fulminate hepatitis model 6h in mice liver, spleen and bone marrow tissues. This demonstrated that MDSCs significantly inhibited the proportion of NK cells.In summary, with the development of acute fulminate hepatitis, Gr1+CD11b+MDSCs cells has rapid proliferation in different organs, at the same time, CD3-NK1.1+NK cells in liver increased. This demonstrated that MDSCs inhibited the increase of the number of NK cells with the development of acute fulminate hepatitis, but not fundamentally reversed the acute fulminate hepatitis and the enhanced activity of NK cells. This project provides a new way of thinking for pathogenesis and treatment of acute fulminate hepatitis.