IL-6 Induce Macrophages Differentiation Into M2-like Phenotype Macrophages In Vitro

BackgroundMacrophages are a group of cells with a variety of characteristics in the human immune system,which play important roles in the prevention of bacterial,viral and parasitic infections.With the deepening of the research,the role of macrophages in the development of tumor has been paid more and more attention.Macrophages infiltrating in the tumor microenvironment are often regard as tumor associated macrophages(TAMs).TAM is a kind of macrophages that differentiated from peripheral mononuclear cells which are recruited by local CCL2,M-CSF,and other cytokines[1].There are two main types of macrophages,M1 and M2 type.M1 type macrophages can be induced by LPS or IFN-γ[2,3],IL-4 and IL-13 can induce macrophage to M2 polarization[2].M1 macrophages mainly produce inflammatory cytokines,like TNF-α and IL-12[4],playing an important role in removalling of bacteria,viruses and fungi and killing microorganisms[5].However,M2 macrophages suppress Th1 inflammatory responses and generate IL-10 and transforming growth factor(TGF-β)[6].M2 type macrophages also have an great effect on tissue remodeling,angiogenesis,parasite infection and tumor progression[7].Interleukin-6 has pleiotropic effects that can affect multiple functions in humans,such as vascular disease,lipid metabolism,insulin resistance[8],and neuropsychological behavior[9].IL-6 is a powerful activator of the STAT3 signaling pathway,activated p-STAT3 protein rapidly translocates to the nucleus,and binding to the recognition sequence of target genes such as cyclin D1,c-myc,and VEGF promoter[10],thereby increasing the transcription and expression of these target genes.A large number of studies have found that IL-6 induced STAT3 activation plays an important role in tumor progression.In previous studies,we found that the expression of IL-6 in gastric cancer tissues was higher than that in non tumor tissues.At the same time,there was an increase in the number of M2 macrophage infiltration in cancer tissues.In order to explore whether IL-6 have an effect on M2 macrophages polarization,we established macrophage differentiation model in vitro.Through detecting the phenotype of the IL-6 induced macrophage,we found that IL-6 could induce normal macrophage differentiation into M2 macrophages,mainly for the increased expression of IL-10 and TGF-β,reduced expression of IL-12.Further more,we explored the mechanism of this inducing differentiation,and function of the induced M2 like macrophages was further studied.ObjectivesTo establish the IL-6-induceing macrophages differentiation cell model in vitro,and to study the effect of IL-6 on macrophage differentiation and the specific signal mechanism.Moreover,to investigate the effect of M2 like macrophages that induced by IL-6 on the biological function of gastric cancer cells.Methods1.The positive Macs separation was used to separate CD14+ monocyte from peripheral blood of healthy adults(PBMC).The purified CD14+ monocyte were stimulated under the recombinant human macrophage colony-stimulating factor(M-CSF)for 5 days for a macrophage differentiation.Futher differentiation of macrophages were induced by IL-6 for 24 h.IL-10 and TGF-β in the differentiated macrophages were detected by quantitative PCR.2.IL-6 can activate the JAK-STAT3 signaling pathway.In this study,we identified that IL-6 could induce the macrophages to M2 type polarization,and in the next study,we detected the STAT3 signal pathway,the activation of this signaling pathway was detected by Westen blot and immunofluorescence.To further study the role of STAT3 in the process of macrophage differentiation in this modle,we used the small interfering RNA(si RNA)gene interference research method,the expression of STAT3 in macrophages was pre-silenced,and then the same quantitative PCR for IL-10 and TGF-β were taken.3.After the study of the phenotype and mechanism of IL-6 induced macrophages,we performed the cell invasion and cell proliferation assay(CCK8)to study the biological function of the induced macrophages.The effects of macrophages on the invasion and proliferation of gastric cancer were studied by co-culturing gastric cancer cells(AGS,SGC-7901)with supernatant from M2 like macrophage that induced by IL-6.Results:1.In the study,it showed that the macrophages which under the stimulat ion of IL-6 for 24 h had an increased expression of IL-10,and an increased expression of TGF-β,but the IL-12p35 expression decreased in the contrary.Moreover,with the increase of the concentration of IL-6,macrophage showed a higher expression of IL-10 and TGF-β,but the expression level of IL-12p35 was decreased with the increase of IL-6 concentration.2.In the study of detecting STAT3 and p-STAT3 by Western-blot,the results showed that in group of macrophages that stimulated with IL-6,the expression of the total STAT3 of the cells did not increase,while the phosphorylated STAT3(p-STAT3)expression increased significantly;By improving the stimulus concentration of IL-6,the total STAT3 expression remained unchanged in cells,while the level of the activated p-STAT3 significantly increased with IL-6 concentration increasing.Immunofluorescence assay showed that the IL-6 stimulated macrophages expressed a large number of activated p-STAT3 protein,and the activated p-STAT3 were major distributed in the nucleus.After STAT3 gene was pre-silenced by si RNA,the results showed that in si RNA silenced cell group,the expression of p-STAT3 decreased.With the decreasing expression of p-STAT3,expression of IL-10 and TGF-β also depressed comparing with non-silenced group.3.Two gastric cancer cell lines(AGS,SGC-7901)were co-cultured with the supernatant of M2 like macrophages.In the migration assay,for both of the two cell lines,the results showed that the number of migrated tumor cells in M2 supernatant groups(AGS 257.6±6.26,SGC 218.6±4.62)were significantly higer than the number in the control groups(AGS 187.8±6.09,SGC 152±7.91).Similarly,the CCK8 results show that the OD450 values at the 72 h time point of gastric cancer cells in the M2 supernatant group(AGS 1.25±0.12;SGC 1.33±0.14)were significantly higher than that in RPMI-1640 control group(AGS 0.90±0.02;SGC 0.98±0.07).Finally,after the addition of IL-10 or TGF-β antibody into the supernatant of M2 macrophages,the pro-migration and pro-proliferation of the supernatant of M2 like macrophages were attenuated.Conclusion:1.IL-6 induces macrophages differentiate into M2 like macrophages with high expression of IL-10 and TGF-βin a dose-dependent manner.2.The activation of STAT3 plays an important role in IL-6 induced M2 macrophages polarization.3.IL-6-induced M2 like macrophages can promote the proliferation and migration of gastric cancer cells,thereby promoting tumor progression.