| Atherosclerosis with its clinical manifestations such as coronary artery disease (CAD), is an underlying cause of myocardial infarction (MI), stroke, and other cardiovascular diseases and has become the leading cause of death worldwide. Emerging evidences support the concept that atherosclerosis is a chronic inflammatory disease, in which innate and adaptive immunity operate together to influence lesion progress. Lymphocytes and monocytes are well known major immune cells responsible for atherosclerosis development. Besides these major cells, there are also many other immune cells, such as dentrtic cells (DCs), Natural killer (NK) cells and mast cells, which are becoming increasingly important in understanding the complexity of this disease process. However, sufficient knowledge to identify the molecules involved in atherosclerosis and develop targeted immunomodulatory strategies has not yet been obtained.T cell immunoglobulin-and mucin-domain-containing molecule-3 (Tim-3), originally identified as a Th1-specific cell surface molecule, has received much attention as negative regulator of Th1 mediated immune response. Engagement with its ligand galectin-9, Tim-3 induces Th1 cells apoptosis and regulates Th1/Th17 cytokine profiles which involve in various inflammatory diseases. Increased body of data have shown that Tim-3 also express abundantly on innate immune cells such as NK cells, macrophages and DCs. However, the effects of Tim-3 on innate immunity remain controversial. The precise roles and exact mechanisms of Tim-3 on innate immunity needs further study. Concerning the critical roles of Tim-3 on T cells and macrophage, the two major cells in atherogenesis, it is reasonable to suppose that Tim-3 may involve in atherosclerosis.In this study, we showed evidence that Tim-3 is upregulated on monocytes and NK cells in AS patients, then studied its roles in the regulation of monocytes/macrophages and NK cells, and verified the effect of Tim-3 on plaque formation in ApoE knockout (apolipoprotein E gene knockout, ApoE-/-) mice. Our work thus provides new insights into mechanism of innate immune regulation by Tim-3 in atherogenesis and also justified the possibility that Tim-3 could serve as a potential target for drug design.PART I The Expression of Tim-3 in AtherosclerosisTim-3 expressed on both adaptive and innate immune cells which were involved in atherosclerosis. In order to evaluate the involvement of Tim-3 in atherogenesis, we first detected the expression of Tim-3 in AS patients and ApoE-/- mice.1. Tim-3 expression is upregulated in organs and aorta plaques from ApoE-/-mouseWe next examined Tim-3 expression in organs and plaques from 14-weeks-old ApoE-/- mice and C57BL/6 mice, which were fed with high diet for 6 weeks. RT-PCR showed higher expression of Tim-3 mRNA in spleens, which contain abundant immune cells. Augment expression was detected in organs of ApoE-/- mice, including heart, liver, spleen and aorta, compared to C57 mice.Tim-3 positive staining was detected in aorta plaques of early stage by immunohistochemistry. It is reported that plaque of early stage purely contains macrophages and T cells. Our results showed that the cells in plaque of early stage were almost all positively stained, suggesting immune cells in plaques expressed Tim-3.2. Tim-3 expression is upregulated on monocytes and NK cells from AS patients and correlated with many etiological and aggravating elements of atherogenesisBlood samples from 97 AS patients, including 24 stable angina pectoris (SAP) patients and 73 unstable angina pectoris patients, which are angiographically documented atherosclerosis, and 51 healthy subjects were collected. There were 24 myocardial infarction patients in the UAP group. Flow cytometry analysis showed that Tim-3 expression was high on CD14+ monocytes and CD16/56+ NK cells, but low on both CD4+ and CD8+ T cells in human PBMCs. Further analysis showed significantly higher expression of Tim-3 on monocytes or NK cells from atherosclerotic patients than that of healthy controls. (Monocytes, p＜0.0001; NK cells,p=0.0005) No significant differences of Tim-3 expression were observed on either CD4+ or CD8+ T cells between the two groups. Atherosclerosis is a multifactor, highly complex disease, in which many etiological and aggravating elements operate together to influence lesion progress. Analysis showed Tim-3 expression on monocytes was correlated well with triglyceride (TG,p=0.0320) and systolic pressure (p=0.0016), while Tim-3 expression on NK cells was positively correlated with TG (p=0.0121) and cholesterol (Cho,p=0.0220)2. The correlation of Tim-3 with inflammation in atherosclerosisEnzyme-linked immunosorbent assay (ELISA) detection showed serum level of C-reactive protein (CRP) and tumor necrosis factor (TNF)-α, the two inflammatory markers, were much higher in AS patients, compared to healthy subjects. (CRP, p= 0.0237; TNF-a, p=0.0235) And Tim-3 is positively correlated with both of them. Further more, Tim-3 expression is higher in patients with more severe inflammation. These results confirmed that Tim-3 positively correlated with inflammatory severity in AS patients.All above results indicated that Tim-3 is involved in atherogenesis through regulating monocytes/macrophages and NK cells.PART II The Regulation of Tim-3 on Monocyts/Macrophages in AtherosclerosisMonocytes/macrophages is one of the most important effector cells in atherogenesis. Recruitment of monocytes from peripheral blood to the intima of the vessel wall is a primordial event in atherogenesis. The results in Part I showed Tim-3 expression was upregulated on monocytes from AS patients and correlated with not only etiological and aggravating elements but also inflammatory severity, strongly supporting the idea that Tim-3 is involved in atherosclerosis. Constitutive expression of Tim-3 was already found on monocytes/macrophages, but the influence of Tim-3 on monocytes/macrophage is still not clear. To reveal the role of Tim-3 on monocytes/macrophages functions, we carried on a series of assay in vitro using the mouse monocytes/macrophages cell line RAW264.7 and purifed peripheral blood monocytes from atherosclerosis patients.1. LPS/ox-LDL induces augment expression of Tim-3 in mouse monocytes/macrophages cell line RAW264.7Lipopolysaccharide (LPS) is recognized as a strong inflammatory inducer, and oxidized low density lipoprotein (ox-LDL) is a etiological and aggravating element of atherosclerosis. Immunocytochemical staining and real-time PCR both showed upregulation of Tim-3 expression on RAW264.7 induced by either LPS or ox-LDL. LPS treatment induced higher expression of Tim-3, approximately twice as much as ox-LDL.2. The regulation of Tim-3 on cell functions in RAW264.7 cellsPeripheral monocytes secrets nitric oxide (NO), a critically important protective signaling molecule of atherosclerosis which clearly has a critical role in the maintenance and repair of the vasculature. After migrating into the vessel wall, monocyte-derived macrophages are turned into fat-loaded macrophages by taking up modified, oxidized or acetylated LDL, residing in the vessel wall and furthering the local inflammatory response by secretion of proinflammatory cytokines, such as TNF-α, interleukin (IL)-1βand IL-6. To further demonstrate the view that whether Tim-3 regulates NO production and proinflammation cytokines secretion in monocytes/macrophages, anti-mouse Tim-3 antibody/Tim-3-Fc (blocking/neutralizing) incubation or pcDNA3-Tim-3 plasmid DNA transfection were introduced in cultured RAW264.7 cells or purified peripheral monocytes before stimulation with LPS or ox-LDL. Supernant were collected for ELISA and Griess assays and cells were used for RNA extraction and RT-PCR analysis.2.1 Tim-3 promotes secretion of proinflammatory cytokines in monocytes/macrophagesELISA and RT-PCR showed that compared to isotype controls, pretreatment of blocking anti-Tim-3 led to decrease of TNF-a production in RAW264.7 cells with or without LPS or ox-LDL treatment. Consistently, a increase was detected in pcDNA3-Tim-3-transfected RAW264.7 cells. Real-time PCR showed the similar effect of Tim-3 on production of IL-1βand IL-6.To further confirm the results in human cells, monocytes were freshly purified from PBMCs of atherosclerosis patients by positive selection using CD14+ microbeads. Cells were blocked using human Tim-3-Fc fusion proteins and then stimulated with LPS or ox-LDL. ELISA. Results showed that Tim-3 blocking decreased TNF-αsecretion, consisting with the results in RAW264.7 cells.These results indicate that induced by LPS or ox-LDL, Tim-3 promotes monocytes/macrophages mediated inflammation.2.2 Tim-3 inhibits NO secretion in monocytes/macrophages Results of colorimetric assay using Griess reagent showed plasma NO levels had no significant differences between 29 AS patients and 16 healthy subject, but NO level in AS patients had increased coefficient of variance (AS vs NOR,1.41 vs 0.74), indicating an imbalance of NO in AS patients. Further more, NO level in AS patients was positively correlated with Tim-3 expression on monocytes. An increase in NO production was detected in anti-Tim-3-treated RAW264.7 cells induced by either LPS or ox-LDL, compared to controls. Consistently, a decrease was detected in pcDNA3-Tim-3-transfected RAW264.7 cells. When induced nitric oxide synthase (iNOS) was analyzed by RT-PCR, similar results were got.To further confirm the results on human cells, monocytes were freshly purified from PBMCs of atherosclerosis patients by positive selection using CD14+ microbeads. Cells were blocked using human Tim-3-Fc fusion proteins and then stimulated with LPS or ox-LDL. The supernatant were collected for NO detection. Results showed that Tim-3 blocking increased NO secretion, consisting with the results in RAW264.7 cells.These results indicate that induced by LPS or ox-LDL, Tim-3 inhibits NO secretion in monocytes/macrophages.3. The signaling pathway in Tim-3-mediated monocytes/macrophages regulationTo study the signaling pathways in Tim-3-mediated monocytes/macrophages regulation, Tim-3 blocking antibody pretreated RAW264.7 were stimulated with LPS and western blot was used to detect expression of critical molecules in I-κB/NF-κB pathway and MAPK(Mitogen-activated protein kinase) pathway, the two classical pathways involved in cytokine and NO secretion.3.1 Tim-3 blockade inhibits I-κB/NF-κB pathway.Tim-3 blockade RAW264.7 cell were stimulated with LPS, and cells were collected at indicated time points. Western Blot showed enhanced phosphorylation of NF-κB p65 at 15min and 30min time points, and degradation of I-κB at 15min time piont in Tim-3 blockade group, compared to the control group, suggesting Tim-3 blockade inhibited I-κB/NF-κB pathway.3.2 Tim-3 blockade inhibits MAPK pathway.Tim-3 blockade RAW264.7 cell were stimulated with LPS, and cells were collected at indicated time points. Western Blot showed enhanced phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular regulatedprotein kinase (ERK) at 5min,15min and 30min time points in Tim-3 blockade group, compared to the control group, suggesting Tim-3 blockade inhibited MAPK pathway.Taken together, Tim-3 expression on monocytes were upregulated by etiological factor of atherosclerosis or mediators of inflammation. The upregulated Tim-3 in turn promotes the production of proinflammatory cytokines and inhibits NO secretion of monocytes. Thus, the increased Tim-3 expression on monocytes from AS patients might promotes atherogenesis.PARTⅢThe Regulation of Tim-3 on NK Cells in AtherosclerosisNatural killer cells have been found in every stages of plaques development, and has been shown to be reduced with a concomitant loss in function in CAD. However, the mechanisms remain unclear. It is reported that Tim-3 induced Thl cell apoptosis by binding with its ligand galectin-9, indicating the mechanism of Tim-3 in cell loss. Since NK cells were recently found to be one of the major cellular sources of Tim-3, we here investigated the expression of Tim-3 on NK cells and assessed its possible roles in NK cells in AS.1. Tim-3 inhibits NK cell activityPathogen infection is related to the risk of AS, and has become the independent risk factor of myocardial infarction. As the first line of the body, NK cells play important roles in defense against pathogens, especially virus, and display their functions by mediating cytotoxicity as well as augmenting immune responses as a result of cytokines production. However, many studies reported NK mediated cytotoxicity damaged vessel cells, and promotes atherogenesis. Thus, the effects of NK mediated cytotoxicity on atherogenesis are multiple. In order to analyze the role of Tim-3 on NK cytotoxicity, Tim-3-Fc fusion proteins were involved in cytotoxic assay in which NK92 cells as effectors and hepatic cell line HepG2 or hepatitis B virus infected HepG2.2.15 as targets. Cell Counting Kit-8 (CCK-8) assay showed that pre-incubation with Tim-3-Fc fusion proteins resulted in an increase in NK92 cytotoxicity and ELISA showed augment production of interferon(IFN)-y, suggesting that Tim-3 could suppress NK-cell functions.2. The regulation of Tim-3 on NK cell loss2.1 Tim-3 expression is negatively correlated with peripheral NK cell quantityConsisting to previous reports, our flow cytometry analysis showed reduced NK cell number in patients with AS compared with healthy controls, (p=0.0016) and significant reverse correlation between Tim-3 expression with NK cell number was found in all AS patients (r=-0.6424,p＜0.0001).2.2 Tim-3 inhibits NK cell proliferationTo verify the correlation between NK cell quantity and Tim-3 expression, we first detected the effect of Tim-3 on NK cell proliferation. We preformed Tim-3 overexpression in cultured NK92 cells by transfecting pcDNA3-human-Tim-3. Results showed Tim-3 overexpression decreased NK92 cell number significantly (p=0.0002), while the percentage of viable cells had no difference between the two group(p=0.7310). Cell cycle analysis revealed the accumulation in G1 phase. These results demonstrated that Tim-3 inhibited NK cells proliferation in vitro.2.3 Tim-3 promotes NK apoptosisThere are many factors influencing the survival of NK cells in AS, among which oxidative stress is particular important. Oxidation of circulating lipids is an important source of oxidative stress. Previous study reported that the susceptibility of NK cells to ox-LDL contribute to the reduced NK cell activity in AS. To verify whether Tim-3 is involved in ox-LDL induced NK cell loss in AS, we blocked Tim-3 pathway with human Tim-3-Fc in NK92 cells treated with ox-LDL. Results showed Tim-3 blockade lead to significant inhibition of ox-LDL-mediated cell apoptosis, (p=0.0078) suggesting Tim-3 might participate in oxidative stress-mediated NK cell loss in AS.Besides ox-LDL, TNF-αis one of cytokines produced by NK cells which plays important role in induction of NK cell apoptosis. Consistent with previous report, our ELISA results showed increased serum TNF-αin patients with AS. Further analysis showed serum TNF-αnegatively correlated with peripheral NK cell percentage (pearson r=-0.3383,p=0.0230). Blocking Tim-3 with human Tim-3-Fc fusion proteins effectively delayed TNF-αinduced NK92 cell death (p＜0.0001). Peripheral NK cells from fresh blood was isolated, and used for the same assay. Results showed Tim-3 blockade decreased TNF-a induced apoptosis of NK cell from AS patients(p=0.0165), but not from healthy subjects(p=0.5958).2.4 Galectin-9 expression of PBMC from AS patients has no correlation with either NK cell quantity or Tim-3 expression on NK cell.PBMCs were isolated from 21 AS patients and 22 healthy subjects. PT-PCR results showed galectin-9 expression had no significant differences between AS patients and healthy subjects. Further analysis showed no correlation with either NK cell quantity or Tim-3 expression on NK cell. Taken together, our data indicated Tim-3 might involve in atherogenesis by inhibiting cytotoxic activity and decreasing cell number through regulating proliferation and apoptosis in NK cells. However, the exact role of NK cells in atherogenesis is still controversial. The studies in different animal models led to different conclusions about depletion of NK cells activity and plaque formation. Thus what's the role NK cell played in atherosclerosis needs further study.PARTⅣEffect of Tim-3 on Plaque Formation in ApoE-/- miceTo verify the effect of Tim-3 on plaque formation in vivo, we inhibited Tim-3 expression in ApoE-/- mice and observed the plaque formation in the aorta.1. lentivirus-shRNA-Tim-3 effectively knockdown Tim-3 expressionRAW264.7 cells were infected with lentivirus-shRNA-Tim-3, lentivirus-shRNA-NS as control. RT-PCR and western blot showed that Tim-3 expression in lentivirus-shRNA-Tim-3 infected cells was decrease approximately 70%, compared to control cells. We next proved the effect in vivo.8-10 weeks old, 18-22g male ApoE-/- mice were fed with high diet for two weeks before injected with lentivirus-shRNA-Tim-3 or lentivirus-shRNA-NS through tail vein. After high fat diet for two months, mice were sacrificed and dissected. RT-PCR showed Tim-3 expression in organs, including heart, spleen and liver, from the ApoE-/- mice injected with lentivirus-shRNA-Tim-3 was much lower than that of control mice.2. Tim-3 gene knockdown decreased plaque formationThese above mice were weighted up at different time points and serum was acquired before sacrificed. The weight and serum TG in the two groups had no statistical difference. We further analyzed aorta plaque formation. Aortas were obtained and stained with Oil red O. Significant decrease of lesion was detected in aorta from lentivirus-shRNA-Tim-3 mice, compared to lentivirus-shRNA-NS mice. These results demonstrated that Tim-3 alleviated atherogenesis while didn't influence body weight and serum lipid level.In summary, this study firstly revealed Tim-3 was upregulated in monocytes and NK cells in AS patients. The augment expression of Tim-3 is positively correlated with inflammation severity in dieases. Blocking assay and overexpression assay showed that Tim-3 promotes proinflamatory cytokines production and inhibits NO production in monocytes/macrophages which are benefit to plague formation. Tim-3 expressed on NK cells suppress NK proliferation and enhance ox-LDL/TNF-a mediated NK apoptosis. Knockdown of Tim-3 in vivo decrease plaque formation in ApoE-/-mice. Our work contributed to the mechanism of immune regulation in atherogenesis and confirmed the idea that Tim-3 could serve as a potential target for drug design for atherosclerosis.
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