A Study Of The Effect Of Interleukin-23on Mouse Bone Marrow Derived Macrophages Polarization

Objective:Macrophages are one of the important components in innate immune system. Macrophages present complex heterogeneity and functional diversity, which are attributed to their distribution, differentiation and the multifarious activating factors. Within a tissue microenvironment, the activation of macrophages is affected by various factors, such as pathogens infection, necrotic cells, and other cytokines, that will lead to the obvious changes in the phagocytosis of macrophages, category of secreted cytokine or chemokine, ability of eliminating tumor and pathogens, and ability of antigen presentation. Our study focuses on the phenotype change of macrophages after cytokine stimulation, and the aim is to find out the effect of IL-23on three M2macrophages (alternatively activated macrophage), which were derived from bone marrow of the C57BL mice, was induced by IL-4, M-CSF and co-culturing with B16F10in vitro, and to investigate the effect of transitional macrophages on tumor cell proliferation and invasion, that will provide a experimental evidence and new way for tumor immunotherapy by targeting on macrophages.Methods:Bone marrow derived macrophages were separated from C57BL mice. Then three M2macrophages were induced by IL-4, M-CSF and co-culturing with B16F10. Upon IL-23exposure, the significant changes of three M2macrophages in morphology, surface receptor, and cytokine expression of three M2macrophages were observed: direct observation of morphology was used by microscope; ELIS Awas used to measure cytokines secreted by macrophages with different stimulations; Western blot and FACs were used to determin the expression of protease in L-arginine metabolism process and specific markers of macrophages respectively. MTT assay and Transwell assay were to evaluate the effect of IL-23treatment group and IL-23itself on proliferation and invasion of melanoma B16F10.Results:Part One: The effect of IL-23for the phenotype of M2macrophages.The results showed that: After the addition of pro-inflammatory cytokine IL-23, a tendency of morphology transformation was visible from "spindle-like" shaped cells to "fried eggs". The levels of six cytokines in macrophage supernatant were determined by using the ELISA, as a result, cytokines category secreted by macrophages changed from IL-10highIL-12lowTNF-αlow to IL-10lowIL-12highTNF-αhigh. The results of western blot showed that IL-23down-regulated the expression of L-arginine metabolism of M2macrophages, and up-regulated the NOS2expression (P<0.01), which was expressed abundantly in Ml macrophages. Flow cytometry showed that IL-23-treated M2macrophages decreased CD206surface expression (P<0.01), and increased the level of MHC-Ⅱ (P<0.01) and CD16/32(P<0.05).Above all, after treated by the IL-23, all the three M2macrophages switched to M1phenotype in the cell morphology, cytokines, metabolism and cell surface marker expression. And the effect of this transition on tumor cells will be further studied next.Part Two:The effect of IL-23treated macrophages on the proliferative and invasive ability of B16F10cells.According to the experimental results of the part one, we selected type M2macrophages supernatant as control group and IL-23treatment group as experimental group to study the impact on B16F10melanoma cell proliferation. Modified Transwell test was used to research the impact on B16F10melanoma cell invasion ability. The results showed that: MTT proliferation assay was used to determine the effects of macrophages conditioned media (CM) on melanoma B16cell growth. M2macrophages could promote the proliferation of tumor cells, while M2macrophages treated by IL-23inhibited proliferation of B16cells (P<0.05). And this effect was associated with TNF-a. Modified Transwell test showed that the transmembrance cells decreased obviously (P<0.01). All results demonstrated that IL-23could switch macrophages phenotype and restore the anti-tumor immune characteristics of M1macrophages. Part Three:Effect of IL-23on proliferation and invasion of B16F10melanoma cellsMost of cytokines exert biological function in a autocrine manner, and local effects on its origin cell or jacent tissue are elucidated. Some study focus on the direct effect of cytokines on tumor cells in proliferation and invasion.In This part Mouse B16melanoma cells were cultured in vitro. Cells in research were treated with and without a certain-concentration IL-23respectively. The results showed that: The growth rate of B16cells treated with IL-23did not show significant change (P>0.05) compared with the control group. Matrigel invasion assay revealed that addition of IL-23to B16cells significantly enhanced its invasive properties (P<0.01), and Gelatin Zymography analysis showed that the MMP-9activity of B16F10cells was significantly increased by IL-23(P<0.01). Conclusions:Above all, the data presented here highlight the effect of IL-23on M2macrophages by Elisa、Western blot、and flow cytometry, as well as the effect of this transtition on B16cell, Themain conclusions are as follows:(1) IL-23can switch M2macrophages to M1, which promote inflammation and exhibit TNF-α、IL-12highIL-10low phenotype, and the iNOS, CD16/32, MHC-Ⅱ are also up-regulated respectively.(2) Durning this switching process induced by IL-23, macrophages can produce some cytotoxicity medium, that can repress the proliferation and invasion of melanoma cells in vitro.(3) There is no significant direct effect of IL-23on the proliferation of B16F10, however, the activity of MMP-9is up-regulated by IL-23, that prometes the invasion of melanoma in vitro.