MiR-16 Regulates Mouse Peritoneal Macrophage Polarization And Affects The Function Of CD4~+T Cells

Objective:To investigate the biological effects and mechanism of miR-16 on the phenotypes of macrophages, and on the activation of co-cultured CD4+T lymphocytes.To explore the important role of miR-16 in the macrophage differentiation process, to provide new potential targets for anti-tumor immune treatment.Methods:1 The regulating role of miR-16 in mouse peritoneal macrophage polarization1.1 100ng/ml IFN-? and 20 ng/ml LPS were used to induce peritoneal macrophages to M1 macrophages;20 ng/ml IL-4 was used to induce peritoneal macrophages to M2 macrophages.CD16/32,IL-12 and iNOS were chosen as the markers of M macrophages;CD206,Dectin-1 and IL-10 were chosen as the markers of M2 macrophages. Surface moleculars CD16/32,CD206 and Dectin-1 were evaluated by flow cytometry.The cytokine levels of IL-12 and IL-10 in the supernatant of cultures were detected by ELISA.The activity of iNOS was determined by Griess method.1.2 LV-miR-16 lentivirus particles were used to infect peritoneal macrophages and M2 macrophages induced by IL-4,surface moleculars CD16/32,CD206 and Dectin-1 were evaluated by flow cytometry.The cytokine levels of IL-12 and IL-10 in the supernatant of cultures were detected by ELISA.The activity of iNOS was determined by Griess method.2 The effects on T cells function when miR-16 regulated M2 macrophage polarization and the possible mechanisms.2.1 Purified CD4+T cells and M2 macrophages infected by LV-miR-16 lentivirus particles were co-cultured in vitro.The activity of CD 69 on CD4+T cells was analyzed by flow cytometry.The cytokine levels of IL-2 and IFN-? of T lymphocytes were evaluated by ELISA.2.2 Mirbase/Targetscans/PicTar were used to predict the potential target mRNA of miR-16.2.3 M2 macrophages induced by IL-4 were infected by LV-miR-16 lentivirus particles and the expression of PD-L1 protein was tested by western blot assay.Results:1.In vitro experiments showed that miR-16 promotes M1 macrophages polarization, and inhibited M2 macrophage polarization.1.1 The results showed that upon IFN-?/LPS stimulation, CD 16/32 expression increased significantly (P<0.05) with no significant changes in CD206 and Dectin-1 expression. Following IL-4 stimulation, CD206 and Dectin-1 expression increased significantly compared to unstimulated cells (P<0.05) while CD16/32 expression did not change.The ELISA assay results indicated that peritoneal macrophages secreted significantly higher levels of IL-12 following IFN-?/LPS stimulation compared to cells without stimulation, but almost with no detectable level of IL-10; however, IL-4 stimulated peritoneal macrophages barely secreted IL-12 but significantly higher levels of IL-10 (P<0.05). Compared to unstimulated peritoneal macrophages, IFN-?/LPS stimulated cells expressed significantly higher level of iNOS(P<0.05).The results suggested that IFN-?(100ng/ml)and LPS (20 ng/ml Successfully induced the alteration of native macrophages to M1-polarized macrophages;IL-4 (20 ng/ml) successfully induced the alteration of native macrophages to M2-polarized macrophages.1.2 Compared to the control group,the expression of CD 16/32 upregulated,with no significant changes in the expression of CD206 and Dectin-1 on the peritoneal macrophages infected by LV-miR-16 lentivirus.The secresion of IL-12 and the activity of iNOS were higher and IL-10 didn't change significantly in contrast with the control group.1.3 Compared to the control group,the expression of CD206, Dectin-1 on the M2 macrophages infected by LV-miR-16 lentivirus dramatically downregulated while the expression of CD16/32 upregulated.The secresions of IL-12 and the activity of iNOS were higher and IL-10 was lower in contrast with the control group.2. miR-16 changed the phenotype of M2 macrophages and affected T cell functions,the possible mechanisms may relate to the regulation of PD-L1 protein expression.2.1 In the co-cultured system,the activity of CD69 in CD4+T cells co-cultured with M2 macrophages overexpressed miR-16 were significantly higher than those in the control groups(P <0.05) and significant higher levels of IL-2 and IFN-? (P<0.05).2.2 Western blotting results showed that the expression of PD-L1 on the M2 macrophages overexpressed miR-16 was significantly lower than the control group.Conclusions:1. miR-16 played an important role in the phenotypes of macrophages,it could promote M1 macrophage polarization, and inhibit M2 macrophage polarization.2. miR-16 could inhibit M2 macrophage polarization to improve the function of T lymphocytes,the possible mechanisms may relate to the regulation of PD-L1 protein expression.