| Objective: Myeloid derived suppressor cells(MDSCs)conclude a group of immature myeloid derived heterogeneous cells that have inhibitory effect on T cell response.MDSCs have suppressive effect on both innate immune responses and adaptive immune responses of immune cells;trigger immune cells in a state of no response or tolerance and can not eliminate mutated precancerous cells in time or kill tumor cells.MDSCs also participate in regulating tumor biological behavior through non-immune mechanisms such as promoting tumor neovascularization,tumor cell invasion and metastasis.The functional importance of these cells in the immune system has been appreciated due to accumulating evidence that has demonstrated their contribution to the negative regulation of immune responses during cancer and other diseases.Modulation of MDSCs may be one of potential cancer therapy methods.Methionine enkephalin(MENK)is an endogenous opioid peptide composed of five amino acid residues,which is characterized by autocrine and paracrine and widely exists in neuroendocrine system.MENK has significant immunoregulation and antitumor effects.By regulating the expression of opioid receptors and binding to opioid receptors,MENK can induce tumor cell cycle arrest,induce cell apoptosis,and have anti-tumor effects on various tumors such as melanoma,glioma,lung cancer and gastric cancer.MENK can also directly improve the killing effect of immune system cells on tumor cells or change tumor microenvironment to play an anti-tumor role by generating immune regulation effect on immune system cells.We have carried out a systematic study on the immunomodulatory effect of MENK in the earlier period,and confirmed that MENK has regulatory effects on T cells,macrophages,NK cells and DCs.However,the modulatory effect and mechanism of MENK on MDSCs are still unclear and need further demonstration.The purpose of this study was to explore the modulatory effect of MENK on murine MDSCs and to investigate the relationship between this modulation and the expression of opioid receptors through experiments both in vitro and in vivo.Material and Methods: About experiments in vitro,famale C57BL/6J mice of 8-10 weeks were used to establish tumor-bearing mice models.Flow cytometry was used to explore the expansion of MDSCs in organs of tumor-bearing mice and normal mice.MDSCs generated from spleen and bone marrow of tumor-bearing mice were isolated by positive immunomagnetic beads.Different doses of MENK stimulated MDSCs in vitro for 48 h,and the proliferation of MDSCs was analysised by CCK8 test to determine the optimal concentration in vitro.The administration of MENK alone or combined with NTX were given to each group of cells for experimental study: Flow cytometry was used to detect ROS content in MDSCs,RT-q PCR was used to analysis the m RNA expression levels of μ and δ opioid receptors in MDCS cells,and ELISA was used to detect the levels of cytokines IL-1β,IL-4,IL-6,IL-10 and TGF-β1 secreted in supernatant of MDSCs.About experiments in vivo,the same tumor-bearing mice models were established as mentioned before.After 14 successive days of MENK treatment,volumes and weights of tumors were detected.Histopathological changes of tumor were observed by H-E staining and immunohistochemistry.Cytokines IL-1β,IL-4,IL-6,IL-10 and TGF-β1 in serum of mice were detected by ELISA.Immunomagnetic beads were used to positively isolate MDSCs and flow cytometry was used to detect the changes of MDSCs and their subpopulations in spleen and bone marrow of tumor-bearing mice.RT-q PCR method was used to analysis the relative expression levels of opioid receptor m RNA in MDSCs and to detect the relative expression levels of MDSCs suppressive function-related molecules such as Arg-1,i NOS,Ets-1and sted1 b.Results: 1.Flow cytometry analysis showed that the content and proportion of MDSCs from spleen and peripheral blood of normal mice were lower than those of tumor-bearing mice(P <0.01).The proportions of MDSCs in various organs of tumor-bearing mice from high to low were: bone marrow,peripheral blood,tumor tissue,spleen,lymph node and thymus.2.The purity of MDSCs and its two subpopulations can be significantly improved by immunomagnetic beads sorting,the purity of MDSCs from spleen > 80%and the purity of MDSCs from bone marrow > 90%.3.MENK inhibited the proliferation of MDSCs in vitro in a dose-dependent manner,with the optimal concentration of 1.25mg/ml.4.Compared with the control group MENK reduced the content of ROS in MDSCs from splenocyte(P <0.05),content of ROS in MDSCs from bone marrow showed a downward trend.5.MENK could up-regulate the expression level of MOR and DOR in MDSCs(P <0.01),NTX could eliminate this effect.6.MENK could stimulate MDSCs from slpenocyte secreting IL-4(P < 0.05),and stimulate MDSCs from bone marrow secreting IL-6(P < 0.05),there was no significant changes in the other cytokines.7.Post treatment with MENK in vivo,weights and volumes of tumors from tumor-bearing mice were significantly reduced(P <0.05).H-E and IHC staining results showed that lymphocyte infiltration in tumor tissue junction of MENK group increased compared with the control group.8.The level of TGF-β1 in serum from tumor-bearing mice decreased while the level of IL-4,IL-6 and IL-10 increased(P< 0.05).9.MENK significantly decreased the proportion of MDSCs from spleen(P <0.05),which the proportion of PMN-MDSCs was decreased(P <0.01)and the proportion of M-MDSCs was increased(P <0.05).The proportion of MDSCs and its subpopulations from bone marrow showed a downward trend.10.MENK increased Arg-1 gene expression level and decreased i NOS gene expression level in MDSCs(P < 0.05).MENK down-regulated the gene expression level of setd1 b and transcription factor Ets-1 in MDSCs(P < 0.01).11.MENK up-regulates the levels of MOR and DOR in MDSCs in vivo(P <0.01),and NTX can completely block this effect in spleen(P <0.01).Conclusion: 1.MDSCs accumulated in tumor-bearing mice.2.MENK could negatively regulate MDSCs of tumor-bearing mice,inhibit cell proliferation,reduce the proportion of MDSCs,and alleviate immunosuppressive function of MDSCs.3.MENK has contrary modulation of the two subpopulations of MDSCs.The reduction of the total MDSCs is mainly achieved by reducing the proportion of PMN-MDSCs subpopulations.4.MENK could increase the expression of μ and δ opioid receptors in MDSCs and reduce the relative expression levels of Ets-1 m RNA and setd1 m RNA of MDSCs,this mechanism may attribute to negative regulation of MDSCs. |
