| BackgroundTim-3(T cell immunoglobulin domain and mucin domain-containing molecule-3) is a newly discovered immunomodulatory molecule, which plays an important role in immunity regulation. As the Thl cell surface marker, Tim-3can bind with its ligand galectin-9and then negatively regulates the function of Thl cell and induces apoptosis of Thl cell. Recent studies have found that the expression level of Tim-3in innate immune cells is also high, including NK cells, macrophages and dendritic cells, and Tim-3involved in the innate immune responses. But the role of Tim-3in innate immune mechanism is still controversial. To expound the effect and mechanism of the Tim-3pathway in the regulation of innate immunity is of great significance to finding the pathogenesis and new therapeutic targets of immune-related diseases.Macrophages, as a necessary member of innate immune, participate in a variety of immune-related diseases. They are the first line of defense in the body fight with infection. Macrophages are plastic, and they can mainly polarize into two types according to different microenvironments:classically activated macrophages (M1) and alternatively activated macrophages (M2). The functions of M1and M2are quite different in the inflammatory responses and tumor development. Many studies have shown that TAMs(Tumor-associated macrophages) are the main groups in the tumor-infiltrating lymphocytes. And It has been confirmed that TAMs are mainly belong to the M2population. TAMs can promote tumor growth by affecting angiogenesis, immune suppression, invasion and metastasis. Science reported in2007that Tim-3expressed on macrophages constitutively and highly, and could promote the inflammatory response of macrophage through NF-kB pathway. At present, there are no studies on whether Tim-3influences macrophage polarization and TAMs function, and thereby affects tumor development. So in this study, we take advantage of clinical specimens, invitro and invivo experiments to explore the expression and function of Tim-3in M1/M2macrophage polarization and in the development of liver cancer, and to provide new ideas for regulating antitumor immune responses.Method1Tim-3expression on the M1/M2macrophages and its regulating role of the macrophage polarization1.1Induce mouse peritoneal resident macrophages and bone marrow cells to M1/M2macrophages respectively, and detect the expression of Tim-3We collected intraperitoneal resident macrophages of3～6mice, and then seeded them in24-well culture plates,1×106cells per well. After24hours, stimulated these macrophages by LPS or IL4with a final concentration of100ng/mL or10ng/mL respectively, and setted PBS control group at the same time. The aim was to induce them into M1/M2macrophages. IL-12and IL-10secretion was detected by ELISA. Molecular markers of M1/M2macrophages was detected by RT-PCR to show whether M1/M2macrophages were induced successfully. And Tim-3expression in each group after24h Stimulation was detected by Flow cytometry.Freshly isolated bone marrow cells were seeded in24-well culture plates,1×106cells per well. And cultured them with20ng/mL GM-CSF or100ng/mL M-CSF for5days respectively, then stimulated them with lOng/mL of LPS for24hours. Collected the cells and their supernatant. Similarly, we detected whether M1/M2macrophages were induced successfully, and analyzed Tim-3expression in each group by flow cytometry.1.2Interference experiments in vitro to investigate the effect of Tim-3on M1/M2macrophage polarizationWe designed and synthesized siRNA oligo for mouse Tim-3and unrelated sequences at the same time. Then we transfected the siRNA oligo and NC oligo into starch induced peritoneal macrophages by Lipo2000. Tim-3expression was detected by RT-PCR, Western blot (WB) to verify that the interfering effect. Peritoneal resident macrophages or bone marrow cells stimulated by the cytokines GM-CSF or M-CSF for60hours, were transfected with Tim-3siRNA oligo and NC oligo by Lipo2000. After48hours, these cells were stimulated by LPS or IL-4respectively to continue the induction of M1/M2macrophages. Twenty four hours later, we collected the supernatant and detected M1/M2characteristic cytokines(IL-12and IL-10) by ELISA.2Tim-3regulates macrophage polarization and impacts on liver cancer cell2.1Tim-3expression on CD14+cells in PBMC and tissues of HCC patientsWe collected24fresh peripheral heparinized blood samples of primary hepatocellular carcinoma (hepatocellular carcinoma, HCC) patients and30blood samples of healthy volunteers. And four cases of primary liver cancer tissues and corresponding cancer adjacent tissues were collected, digested3-6h by collagenase, and grinded gently on200mesh stainless steel net. We then collected the single cell suspension carefully. The infiltrating lymphocytes of HCC tissues were isolated using Ficoll lymphocyte separation medium. These cells were stained by CD14, Tim-3and the corresponding fluorescence isotype control antibodies at the same time, and then Tim-3expression on CD14+cells was detected by flow cytometry.2.2Coculture experiment to study liver cancer cell-mediated macrophage polarization as well as Tim-3expression and regulationWe cocultured the starch-induced mouse peritoneal macrophages with H22in a24-well culture plate in a ratio of one to four, and setted the time point:12h,24h,48h. After washing the suspending H22, collected the adhering macrophages and detected Tim-3expression by flow cytometry. Molecular markers of M1/M2macrophages was detected by RT-PCR.2.3The influence of blocking Tim-3on liver cancer cell-mediated macrophage polarizationFreshly isolated mouse peritoneal macrophages were seeded in24-well plates, at a density of5×104/mL,500mL per well. After the macrophage cells adhered, we treated them with a final concentration of5μg/mL Tim-3blocking antibody or IgG control for1hour, then we added2×105H22cells to form the co-cultur system. Forty eight hours later, the peritoneal macrophages were collected, and the M1cytokine marker IL-12was detected by flow cytometry. 2.4Animal experimental studies of Tim-3-mediated macrophage polarization in tumorigenesis6-8weeks-old male BALB/c mice were selected and injected with1×106mouse hepatoma cell lines of H22in hind limb medial subcutaneously to construct a mouse hepatoma model. After two weeks the tumors were separated, digested with collagenase. Tumor infiltrating lymphocytes were isolated using Ficoll lymphocyte separation medium. At the same time, the corresponding mouse spleen cells were separated too. These cells were labeled by flow cytometry antibody CD11b, Tim-3and the corresponding fluorescence isotype control antibodies, and the Tim-3expression on CD11b+cells was detected by flow cytometry.Bone marrow cells were induced by M-CSF for60hours and then transfected with Tim-3SiRNA oligo by Lipo2000. NC control group was set at the same time. After a continued induction for48h, threefold number of H22cells were added to these macrophages, and injected into BALB/c subcutaneously to construct a mouse hepatoma model. Tumor size was measured on a regular basis, and the tumor growth curve was to calculated.Results1In vitro experiments showed that Tim-3was highly expressed on M2macrophages, and promoted M2macrophage polarization1.1Tim-3expression level of M2macrophages was significantly higher than M1macrophagesTo explore the Tim-3expression in different subsets of macrophages, we respectively used intraperitoneal residence of macrophages and bone marrow cells to aquire M1/M2macrophages. ELISA results showed that the intraperitoneal resident macrophages stimulated by LPS and bone marrow cells induced by GM-CSF secreted a higher level of IL-12(235.01±4.86pg/mL,542.85±5.98pg/mL) and a lower level of IL-10(32.54±3.10pg/mL,87.64±6.26pg/mL); whereas the intraperitoneal resident macrophages stimulated by IL-4and bone marrow cells induced by M-CSF secreted a high level of IL-10(413.49±3.65pg/mL,759.63±5.38pg/mL) and a lower level of IL-12(36.74±3.05pg/mL,186.17±3.10pg/mL), suggesting that, M1/M2macrophages were induced successfully. RT-PCR detection of M1/M2macrophage molecular markers is further evidence of the successful model induction. Tim-3expression on different macrophages were analyzed by Flow cytometry. Results showed that Tim-3expression of M2macrophages was significantly higher than that of M1macrophages (peritoneal macrophages of52.27%±7.02%vs35.53%±1.76%, p<0.05; bone marrow cells60.10%±3.05%vs98.03%±1.56%, p<0.01).1.2Inhibiting the expression of Tim-3can effectively promote the differentiation of M1macrophages and suppress the M2macrophage differentiationTo further study the role of Tim-3in macrophage polarization, we designed Tim-3siRNA oligo and transfected them into macrophages by liposome. RT-PCR and Western Blot results showed that Tim-3siRNA could effectively inhibit the expression of Tim-3. Intraperitoneal resident macrophages and bone marrow cells were collected and cultured, following transfection of Tim-3siRNA. Then these cells were continued to be inducted into M1/M2macrophages. ELISA results show that, compared with the control group, Tim-3siRNA transfection group of intraperitoneal resident macrophages induced by LPS and GM-CSF-induced bone marrow cells’ secreting IL-12levels were significantly increased. Intraperitoneal resident macrophages:250.45±24.60pg/mL vs.587.41±20.63pg/mL, p<0.01; bone marrow cells:423.81±30.82pg/mL vs952.99±177.6pg/mL, p<0.05. In line with this, compared with the control group, Tim-3siRNA transfection group of IL-4stimulated resident macrophages and M-CSF-induced bone marrow cells secreted a significantly higher level of IL-10. Intraperitoneal resident macrophage cells:350.37±11.77pg/mL vs.136.91±5.66pg/mL, p<0.01; bone marrow cells:792.43±68.51pg/mL vs.318.74±56.51pg/mL, p<0.05. All the results indicated that inhibiting the expression of Tim-3could effectively promote the differentiation of M1macrophages, however, inhibit the M2macrophage differentiation.2Tim-3promotes M2macrophage polarization and suppresses the growth of hepatocellular carcinomaReported in many papers, TAMs in a variety of tumors, including hepatocellular carcinoma tumor, mainly belong to the M2type, and usually associate with a poor prognosis. To clarify the liver cancer microenvironment effects on macrophage polarization is of great significance. Our first part of the study results suggested that Tim-3involved in regulating M1/M2macrophage polarization. So in the second part, we focused on Tim-3regulation on macrophage polarization in liver cancer and its impact on liver cancer.2.1Tim-3expression on CD14+cells in peripheral blood and tumor of primary liver cancer patients were significantly increasedTo further study the role of Tim-3on regulating TAMs in liver cancer, we collected peripheral blood samples and liver cancer tissues of hepatocellular carcinoma patients, and analyzed Tim-3expression by flow cytometry. The results showed that, the Tim-3expression on peripheral blood CD14+cells of HCC patients was significantly higher than healthy volunteers (P<0.01). Compared with macrophages in adjacent tissues, Tim-3expression level of TAMs in hepatoma tissues was significantly higher (P<0.05). The results indicated that Tim-3involved in liver cancer TAMs polarization.2.2Hepatoma cells co-cultured in vitro promoted M2macrophage polarization and upregulated the expression of Tim-3We further used the co-cultured invitro system to illustrate Tim-3function of TAMs polarization in liver cancer. The starch-induced mouse peritoneal macrophages were co-cultured with H22in a1:4ratio, and then Tim-3expression at different time points were detected by flow cytometry. FCM results showed that along with the incubation time extend, the Tim-3expression on peritoneal macrophages increased. RT-PCR results showed that the macrophages tended to the M2polarization, and further suggest that Tim-3involved in the formation of liver cancer TAMs.2.3Blocking Tim-3can significantly weaken the hepatoma cell-mediated M2macrophage polarizationTo investigate the influence of Tim-3on liver cancer cell-mediated macrophage polarization, we blocked the Tim-3of co-culture system using a specific monoclonal antibody. Flow cytometry results showed that, after blocking the Tim-3, the ability of macrophages in the secretion of IL-12enhanced (49.80%±1.70%vs.30.10%±1.84%, p<0.05). The results indicated that blocking the Tim-3can significantly weakened the capability of liver cancer cells-mediated M2polarization.2.4Tim-3expression was significantly increased on macrophages of The tumor-bearing mice, and interfering Tim-3in M2could inhibit the tumor growthTo further study the influence of Tim-3-mediated macrophage polarization in tumor development, we constructed the H22subcutaneous tumor-bearing mice model. After two weeks, the largest tumor diameter was up to1cm. The flow cytometry results showed that the Tim-3expression level on tumor infiltrating CD14+cells was significantly higher than spleen CD14+cells of tumor-bearing mice (P<0.001), consistent with the invitro results. Then we transfected M2macrophages with Tim-3siRNA and NC oligo respectively, mixed them with H22, and constructed the H22subcutaneous tumor-bearing mice model. Tumor growth curve and tumor weight analysis showed that the Tim-3interfering group of tumor size and weight were significantly lower than NC control group, suggesting that inhibition of Tim-3expression in M2could significantly suppresses tumor growth.ConclusionsTim-3was highly expressed on M2macrophages, and promoted M2macrophage polarization; Tim-3expression on CD14+cells in peripheral blood and tumor of primary liver cancer patients were significantly increased; Tim-3played an important role in hepatoma cell-mediated macrophage polarization; Inhibition of Tim-3expression in M2could significantly suppress the tumor growth of tumor-bearing mice model.Innovation and SignificanceIn this study, we first demonstrate how Tim-3regulates macrophage polarization and impactes on hepatocellular carcinoma. Our findings can not only help to explain the Tim-3regulation mechanism of innate immunity, but also provide new ideas and new targets to hepatocellular carcinoma immunotherapy. |
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