Study On Role And Mechanism Of Tim-3 On Immune Escape In Benzene-induced Acute Myeloid Leukemia

Part ? Establishment and characteristics of animal model in benzene-induced acute myeloid leukemiaObjectiveBenzene is a chemical substance widely found in people's production and living environment.It can be produced by decoration release,occupational exposure,tobacco burning,automobile exhaust,etc.It is currently a recognized important environmental factor that can induce leukemia,especially acute myeloid leukemia(AML).Long-term chronic benzene exposure primarily damages the hematopoietic system,causing leukopenia,pancytopenia,aplastic anemia and even leukemia.Benzene exposure has been associated with increased incidence of leukemia in humans,in particular acute myeloid leukemia(AML),predominantly based on evidence from epidemiology.Leukaemia treatment costs are higher,the occurrence of benzene leukaemia causes harm to the society.The mechanism of benzene is not fully clear and the corresponding animal models are deficient.Therefore,demonstrating systematically the relationship between benzene and leukemia has important social and scientific value.The method of subcutaneous injection of benzene was simple and easy to operate and closer to the actual benzene exposure of peoples.This study intended to establish the animal model of benzene induced AML by subcutaneous injection of benzene,so as to provide a kind of animal model and further research on the key genes and pathways in benzene-induced leukemia.Methods1.Animal model establishmentSubcutaneous injection of benzene corn oil(the mixing ratio of benzene and corn oil was 1:9)mixed solvent(pure benzene concentration of 250mg/kg·d)was administered to the toxic group for 6 days per week,and rest for 1 day to establish the animal model of benzene-induced acute myeloid leukemia.The control group was given subcutaneous injection of the same dose of corn oil.2.Blood routine testPeripheral blood was collected after anesthetized in the toxic group and control group at different time points.After diluted 10 times with sodium citrate,the blood cell counts of the two group were measured by the five-class blood cell analyzer to observe continuously the changes in the number of white blood cells,red blood cells and platelets.3.Peripheral blood smearThe toxic group and control group were anesthetized with peripheral blood at different time points,and then blood smears were prepared for Wright-Gimsa staining to observe the cell morphology.4.Bone marrow smearThe toxic group and control group were sacrificed after anesthesia,and the femur bone marrow samples were prepared for bone marrow smear.After rushing out,they were naturally air-dried,and then stained with Wright-Gimsa to perform bone marrow cytology examination.5.Histopathological examinationThe toxic group and control group were sacrificed after anesthesia,and the heart,liver,spleen,lung,kidney and bone marrow tissues of the mice were taken,fixed with 4%paraformaldehyde,and paraffin sections were routinely prepared,HE staining and microscopic examination.Results1.Mouse survival statusIn the early stage of exposure,some mice began to be irritated,their spirit were slightly hyperactive,and their weight gain was slow.With the prolonged benzene exposure time,the toxic group mice lost weight,and some of them showed gait instability,vertical hair,arched back,rough skin,hair removal and other similar symptoms.During the benzene exposure stages,some mice in the toxic group developed dyscrasia and died.The mice in the control group grew normally,and no similar results were observed.2.Peripheral blood cell countAt different stages of benzene exposure,the number of peripheral blood leukocytes in the toxic group decreased at the start of stages and then increased at the leukemia outbreak stage.Compared with the control group,the peripheral blood leukocyte numbers significantly increased in the toxic group at 6 months.The number of red blood cells and platelets in the toxic group showed a gradual downward trend.Compared with the control group,the numbers of peripheral blood erythrocyte and platelets significantly decreased in the toxic group at 6 months.3.Blood smearThe blood smear of the mice in the toxic group at 6 months showed the characteristics of rodent blood smear.Ring-shaped neutrophil mononuclear cells,polychromatic red blood cells,platelets and leukemia cells were observed in the blood smear.No leukemia cells were seen in the control group at 6 months in the blood smear.4.Bone marrow smearThe bone marrow smear in the toxic group at 6 months showed that the leukemia cells clustered in the bone marrow,the cell body was large,the proportion of nucleoplasm was imbalanced,and the proportion of original cells was more than 30%.Cytochemical staining showed MPO staining was negative or weak positive,PAS staining was negative,consistent with AML characteristics.The study indicated that AML animal model had been successfully established after six months by benzene exposure.No obvious abnormalities were observed in the control group at 6 months.5.Histopathological examinationIn the toxic group at 6 months,the normal tissues of the lungs were destroyed,and the intrinsic structure of the alveoli was replaced by diffuse and proliferating tumor cells,the morphology of the tumor cells was basically the similar,the cell bodies were large,the nuclei were round and irregular,the nuclear chromatin was loose,and the mitotic images were more common.The normal tissues of the spleens in the toxic group at 6 months were destroyed,and the intrinsic structure was replaced by diffuse and proliferating tumor cells.The leukemia cells were diffusely infiltrated in the spleen and red pulp.The bone marrow tissue proliferation in the toxic group at 6 months was abnormally active,tumor cells were found to aggregate.In the toxic group at 6 months,tumor cells of different sizes and shapes can be seen in the liver portal area,and mitotic images can be seen.There were no obvious abnormalities in the pathological examination of lungs,spleens,bone marrows and livers in the control group at 6 months.There were no obvious abnormal changes in the heart and kidney pathology of the toxic group and the control group at 6 months.ConclusionsExperimental studies have shown that AML can be induced by subcutaneous injection of benzene,and the animal model of benzene-induced AML can be successfully established at 6 months.Benzene exposure affects the weight gain of mice.The effect on the nervous system was first stimulated and then inhibited.After benzene exposure,the numbers of peripheral blood leukocytes decreased in the early stages and increased in the late stage,blood platelets and red blood cell counts decreased.gradually.Poisoning process firstly appeared benzene poisoning performance,finally resulted in acute leukemia.Bone marrow hematopoietic function was inhibited.More primitive cells were observed in bone marrow smear in the toxic group at 6 months.Pathological examination of bone marrow in the toxic group at 6 months showed abnormal proliferation of bone marrow tissue,obvious extramedullary tumor cell infiltration was also observed in the spleen and liver tissues in the toxic group at 6 months.In addition,infiltrating tumor cells were observed earlier in lung tissue in the benzene-induced AML animal model.Part ? Expression and immunosuppressive effect of Tim-3 in benzene-induced AMLObjectiveBenzene is an important environmental pathogenic factor in acute myeloid leukemia(AML),but its specific mechanism remains unclear.At present,there are many researches on the pathogenesis of leukemia,and its final occurrence is closely related to the uncontrolled proliferation of leukemia cells,the inhibition of apoptosis,the clonal proliferation caused by differentiation disorders,and the tumor immune escape caused by immunodeficiency.In the first experiment,the animal model of benzene-induced AML better simulated the changes of hematopoietic microenvironment in the process of benzene leukemia,and provide an ideal animal model for further study of the pathogenesis of benzene-induced leukemia.In the tumor microenvironment,tumor cells,fibroblasts,and endothelial cells can recruit monocytes as the precursor cells of tumor-associated macrophages(TAMs)to the tumor site by means of producing various chemokines.Immunomonitored M1-type macrophages are "domesticated" to immunosuppressive M2-type macrophages,this process is called macrophage polarization.While TAMs secrete various growth factors such as transforming growth factor-?(TGF-?)to promote tumor growth,which is associated with tumorigenesis and poor prognosis.The inhibitory effect of negative costimulatory molecules(also known as negative immunological checkpoint molecules)on immune response has attracted extensive attention as an important mechanism of tumor immune escape.Tim-3(T cell immunoglobulin domain and mucin domain-3)which full name of T lymphocyte immunoglobulin domain and mucin domain-3,as an immunomodulatory molecule,has been extensively studied in the field of immunology.However,the study of its role or expression in blood malignant tumors is limited,especially in the study of benzene leukemia.In view of the highly malignant biological characteristics of leukemia and it is different from other solid tumors,it is necessary to study the expression and significance of Tim-3 in AML induced by benzene.In order to elucidate the pathogenesis of benzene-induced leukemia,monitor and intervene the hematological toxicity of benzene,we will further investigate the immunosuppressive effects in the microenvironment of benzene-induced AML through the expression and function of negative immunological checkpoint molecule Tim-3.This will provide the laboratory basis and immunological targets for the treatment of benzene leukemia.Methods1.Immunocytokine detectionThe peripheral blood in the successfully modeled toxic model group(benzene leukemia model group)and control group was taken from the orbit,separated from the serum,and operated according to the instructions of the Elisa kit.The levels of serum IL-12 and TGF-?1 in the toxic model group and the control group were measured by the microplate reader at 450 nm.2.Flow cytometryThe expression levels of CD8 T cells and CD14+monocytes in the bone marrow,spleen and peripheral blood of the toxic model group and the control group were detected.3.ImmunofluorescenceImmunofluorescence assay was used to detect the expression of Tim-3 on F4/80(M1+M2)macrophages of bone marrow tissues and CD206(M2)macrophages in F4/80(M1+M2)macrophages of bone marrow tissues.Immunofluorescence assay was used to detect the expression of CD 163(M2)macrophages in CD68(M1+M2)macrophages of spleen tissues.Results1.Immunocytokines detect of IL-12 and TGF-?1(1)Cytokine IL-12Serum levels of IL-12 in the toxic model group were significantly lower than that in pre-benzene exposure,(128.65±62.06)pg/mL and(240.46±130.98)pg/mL respectively(p<0.05).Serum levels of IL-12 in the toxic model group significantly reduced than that in the control group,(128.65±62.06)pg/mL and(216.71±70.16)pg/mL respectively(p<0.05).(2)Cytokines TGF-?1Serum levels of TGF-?1 in the toxic model group were significantly higher than that in pre-benzene exposure(103.59±36.13)pg/mL and(54.03±21.07)pg/mL respectively(p<0.05).Serum levels of TGF-?1 in the toxic model group increased significantly than that in the control group,(103.59±36.13)pg/mL and(76.84±15.73)pg/mL respectively(p<0.05).2.Flow cytometry was used to detect the expression levels of Tim-3 on CD8+T cells and CD14+monocytes in bone marrow,spleen and peripheral blood(1)Expression of Tim-3 in bone marrow was detected by FCWThe proportion of Tim-3 positive CD8 T cells and CD 14 monocytes of bone marrow in the toxic model group were significantly higher than that in the control group(p<0.05).(2)Expression of Tim-3 in spleen was detected by FCWThe proportion of Tim-3 positive CD8 T cells and CD 14 monocytes of spleen in the toxic model group were significantly higher than that in the control group(p<0.05).(3)Expression of Tim-3 in peripheral blood was detected by FCWThe proportion of Tim-3 positive CD8 T cells and CD 14+ monocytes of peripheral blood in the toxic model group were significantly higher than that in the control group(p<0.05).3.Immunofluorescence analysis of the expression of Tim-3 in bone marrow macrophages and polarization in bone marrow and spleen macrophagesCompared with the control group,the expression of Tim-3 on F4/80(M1+M2)macrophages of bone marrow in the toxic model group significantly increased.The expression of CD206 macrophages in F4/80(M1+M2)macrophages of bone marrow in the toxic model group significantly increased than that in the control group.The expression of CD 163 macrophages in CD68(M1+M2)macrophages of spleen in the toxic model group significantly increased than that in the control group.Conclusions1.Tim-3 as an immunosuppressive molecule was highly expressed on CD 14+monocytes in bone marrow,spleen and peripheral blood in toxic model group,and the Tim-3 expression level of bone marrow macrophages in the toxic model group was also significantly higher than that in the control group.The results showed that benzene exposure can promote the expression of Tim-3 in macrophages.2.In the benzene-induced toxic model group,with the up-regulation of Tim-3 expression,the serum level of IL-12 as type M1 macrophage functional marker in macrophages decreased,and the serum level of TGF-?1 as type M2 macrophage functional marker increased.The expression of M2 macrophages in the bone marrow and spleen macrophages in the toxic model group was significantly higher than that in the control group,suggesting that benzene exposure can induce the macrophages polarization to type M2.3.The expression level of immunosuppressive molecule Tim-3 on CD8 T cells of the bone marrow,spleen and peripheral blood in the toxic model group also significantly increased,indicating that benzene exposure can promote the expression of negative regulatory molecules on the surface of immune T cells and inhibit T Cell function.That also illustrated the role of immunosuppression in the pathogenesis of benzene leukemia.Benzene-induced AML can up-regulates Tim-3 expression on a variety of immune cells.The study showed that the pathogenesis of benzene-induced AML was involved comprehensive mechanism such as modulating macrophage polarization,reducing the secretion of pro-inflammatory cytokines,promoting the release of anti-inflammatory cytokines,changing macrophage phenotype and function and promoting T cell function depletion,inducing negative immune response in the tumor microenvironment,inhibiting the immune response,thus causing immune escape.Therefore,Tim-3 may be one of the mechanisms that mediating tumor immune escape,which may serve as a marker of benzene leukemia onset and prognosis,and may guide the treatment of benzene-induced AML in the future.Part ? Study on mechanism of up-regulation of Tim-3 and PI3K/AKT/mTOR signaling pathway in benzene-induced AMLObjectiveThe second experiment of this study suggested that the hematological toxicity of benzene may up-regulate the expression of Tim-3 through macrophage polarization,reduction of pro-inflammatory cytokine secretion,promotion of anti-inflammatory cytokine release,changing macrophage phenotype and function The body's immune system gradually produced immunosuppression and tumor immune escape,so increased the risk of leukemia.How did Tim-3 play the role in the process of benzene-induced leukemia?What factors may account for its up-regulation?Studies found that Tim-3 was highly expressed in human acute myeloid leukemia(AML)cells,triggered growth factor-like effect by activating the PI3K/AKT/mTOR signaling pathway,promoted hypoxia-induced neovascularization and enhanced the glycolysis ability of tumor cells by activating HIF-1 signaling pathway,thereby to maintain the survival and proliferation of tumor cells.At the same time,several studies had also confirmed that the PI3K/AKT/mTOR signaling pathway was involved in the proliferative development and activation processes of macrophages and promoted the conversion of macrophages from type M1 to type M2.Therefore,this study used the changes of Tim-3 on monocytes/macrophages in benzene-induced AML as an entry point to further explore the regulatory relationship between Tim-3 and PI3K/AKT/mTOR signaling pathways in benzene-induced AML.The TGF-?/Smad pathway proteins were detected synchronously.From the perspective of the mechanism that Tim-3 mediates the polarization of tumor-associated macrophages and escapes immune surveillance so as to prompt the pathogenesis of benzene leukemia,to further provide theoretical basis for Tim-3 and PI3K/AKT/mTOR signaling pathway as potential targets for benzene-induced AML immunotherapy.Methods1.The expression of PI3K,AKT and mTOR protein in bone marrow tissues was detected by immunohistochemistry in the toxic model group and the control group.2.The expression of p-PI3K,p-AKTP and p-mTOR protein in bone marrow macrophages was detected by immunofluorescence assay in the toxic model group and the control group.3.The expression of TGF-?1 and Smad3 protein in bone marrow tissues was detected by immunohistochemistry in the toxic model group and the control group.Results1.Expression of PI3K/AKT/mTOR pathway protein(1)Immunohistochemical detection of PI3K/AKT/mTOR in femur bone marrowThe results showed that compared with the control group,the expression levels of PI3K,AKT and mTOR protein of the bone marrow in the toxic model group significantly increased(p<0.05).(2)Immunofluorescence detection of p-PI3K,p-AKTP and p-mTOR in bone marrow macrophagesThe results showed that compared with the control group,the expression levels of p-PI3K,p-AKT and p-mTOR of bone marrow macrophages in the toxic model group all significantly increased.2.Expression of TGF-?/Smad pathway proteinsThe results of immunohistochemistry showed that compared with the control group,the expression levels of TGF-?1 of the bone marrow in the toxic model group significantly increased(p<0.05).Compared with the control group,the expression levels of Smad3 of the bone marrow in the toxic model group significantly increased(p<0.05).ConclusionsBased on the preliminary experiment,we further studied the effect of Tim-3 regulation on the immune escape function of tumor macrophages during benzene-induced AML,and then explored the up-regulation of Tim-3,the expression of PI3K/AKT/mTOR signal pathway proteins in bone marrow tissue macrophages increased.So this signaling pathway protein may be involved in the activation process of macrophages,and promote the polarization of macrophages from M1 type to M2 type and that led to AML.At the same time,the expression of TGF-?/Smad pathway proteins of bone marrow in the toxic model group were also significantly higher than that in the control group.So these signaling proteins may also participate in the pathogenesis of benzene leukemia.This results layed important foundation for searching effective targets of benzene leukemia and revealing the key signal molecules and functional regulation networks of upstream/downstream of Tim-3 expression in benzene-induced AML.