Effects And Molecular Mechanisms Of UCP-1 Deficiency On The Activities Of MDSCs And NK Cells

Uncoupling protein 1(UCP-1)is an important member of the uncoupling protein family,involved in thermogenesis,glycolipid metabolism,oxidative stress,inflammation,and tumorigenesis.As an important protein involved in mitochondrial metabolism,the key role of UCP-1 in immunoregulation and molecular mechanism is largely unclear.Based on the UCP-1-/-mice,our group found that the frequency of MDSCs in UCP-1-/-mice spleen,blood and liver is up-regulated and their functions are enhanced.In addition,there was no significant change in the development of NK cells,but a difference in the distribution of NK cells in the peripheral tissues and decreased NK cells function.Thus,the effects of UCP-1 deficiency on the function of MDSCs and NK cells and their molecular mechanisms need to be clarified.This study aims to conduct a preliminary analysis on the effects and significances of UCP-1 deficiency on MDSC and NK cells and explore the role and molecular mechanisms of UCP-1 in tumors and non-alcoholic fatty liver diseases(NAFLD).Our study can be divided into two sections.Part ?.Variations of MDSCs in the UCP-1-/-mice and their Sensitivity to Transplanted TumorObjective:To analyze the variations of frequency and functions of MDSCs in UCP-1-/-mice and detect the sensitivity of UCP-1-/-mice to transplanted tumors.To analyze the preliminary molecular mechanisms of variations in frequency and functions of MDSCs in UCP-1-/-mice.Methods:1.Variations of frequency and subtypes of myeloid immune cells in thymus,bone marrow,spleen,liver and peripheral blood were analyzed by flow cytometry in UCP-1-/-and wild-type mice.2.Expression of effector molecules(iNOS,Arg-1,ROS,PD-L1,and IL-10)by MDSCs were detected by Western blot or flow cytometry.In vivo tumor-transplanted model was used to compare the sensitivity of the mouse to tumor growth.3.MDSCs functions were measured by proliferation,death and glycolipid metabolism.1)MDSCs were labled with CFSE to analyze their proliferation,then the signal molecules involved to MDSCs proliferation,such as p-NF-?B,and p-c-Myc,were determined by Western blot.Also,bone marrow cells were separated to be induced to MDSCs in vitro.2)Apoptosis of MDSCs were identified in vitro by the detection of the signaling molecules of apoptosis,including Caspase3,Caspase8 and Bcl-xl,molecules of pyroptosis,such as Caspasel,Caspasell and GSDMD were detected by Western blot.3)Levels of free fatty acid,triglycride and cholesterol in the UCP-1-/-mice serum were analyzed by ELISA.Variations of mitochondrial mass as well as CD36 were detected by flow cytometry.Molecules associated with the glycolipid,including Glut1,HK ?,CPT-1,ACC were tested by Western blot.Results:1.There were increased frequencies of MDSCs in spleen,liver and peripheral blood of the UCP-1-/-mice,but no significant changes of MDSCs in bone marrow.Compared with PMN-MDSCs,M-MDSCs were increased in the UCP-1-/-mice.2.Expression of Arg-1,iNOS,PD-L1,and IL-10 by MDSCs were similar in WT and UCP-1-/-mice,but ROS was lower in UCP-1-/-MDSCs.The transplanted colon cancer grew faster and larger in UCP-1-/-mice.3.UCP-1-/-MDSCs showed more proliferation with increased p-NF-?B and p-c-Myc.At the same time,there were no significant differences in pyroptosis-associated molecules,including Caspase1,Caspase11 and GSDMD,but Bcl-xl with anti-apoptosis activity was increased,and expressions of Caspase3 and Caspase8 did not vary sharply in UCP-1-/-mice.Besides,there were no changes in serum triglycerides and free fatty acids,but decreased cholesterol in sera' of UCP-1-/-mice.The mitochondrial mass and ACC of UCP-1-/-MDSCs was decreased,but glycolytic enzymes(Glutl and HKII)were increased.In addition,there were no significant changes of CD36 and CPT-1 in UCP-1-/-MDSCs.Conclusions:1.Frequencies of MDSCs(mainly M-MDSC)were increased in spleen,liver and peripheral blood in UCP-1-/-mice.2.UCP-1-/-mice display more sensitivity to transplanted tumors,could be related to the increased MDSCs in frequencies.3.Increased MDSCs in UCP-1-/-mice is involved with increased proliferative capacity and decreased death.4.The expression changes of glycolipid metabolism in MDSCs suggest that metabolic variation may be related to their functions.Part 2.Variations of NK cells in the UCP-1-/-mice and their Sensitivity to Non-alcoholic Fatty Liver DiseaseObjective:To analyze variations in the distribution and function of NK cells in in UCP-1-/-mice and observe the sensitivity of UCP-1-/-mice to non-alcoholic fatty liver disease.Methods:1.Frequencies,NKG2D expression,and IFN-y productionin of NK and NKT cells in the spleen,liver,peripheral blood,and thymus in UCP-1-/-mice were detected by flow cytometry.2.The pathological changes of fatty livers in the non-alcoholic fatty liver models was analyzed by the histological oil-red staining.Tissue distribution,NKG2D erpression,and IFN-y prodiction of NK cells and the expression of NKG2D and in non-alcoholic new fatty liver models was detected by flow cytometry.3.The metabolism-related molecules(mTOR)in CD3-NK1.1+ cells were measured by Western blot and intracellular ROS of in NK cells was detected by flow cytometry.Results:1.Under physiological conditions,there was a decreased frequency of NK cells in liver,but no differences in the spleen and peripheral blood in UCP-1-/-mice.Also,there were no differences in the NKG2D expression and IFN-y prodiction by NK cells of UCP-1-/-mice.2.There was aggravated progress of non-alcoholic fatty liver disease in UCP-1-/-mice induced by the MCD diet.There was obvious fatty deposition and lipid infiltration in UCP-1-/-mice.NK cells and NKG2D-NK cells in UCP-1-/-mice were significantly reduced in spleen,peripheral blood and liver.3.ROS in NK cells was reduced in UCP-1-/-mice and the mTOR was lower in UCP-1-/-NK cells.Conclusions:1.Liver NK cells of UCP-1-/-mice is decreased physiologically.NK cell function is down-regulated in mice with non-alcoholic fatty diseases.2,UCP-1-/-mice display more sensitivity to the development of NAFLD.Whether the downregulated NK cells contribute to the aggravated NAFLD needs further study.