The Mechanism Of Inhibition Of Macrophage Immune Activity By Extracellular Arginine Depletion In Malignant Mammary Epithelial Cells

Background and Objective:The concept of the tumor microenvironment has attracted much attention in recent years.The tumor microenvironment usually has immunosuppression.Although T cells are the main effector cells,macrophages,which account for the largest proportion of immune cells,can not be ignored for their influence on the occurrence and development of tumors and even the anti-tumor immunity of the body in the later stage.Tumor cells often change their metabolic pathways to maintain their growth and proliferation due to excessive nutritional requirements.Similarly,changes in metabolites outside the cell can also affect the activity and function of immune cells,and this process is not overnight.Remodeling the tumor microenvironment is an effective means of cancer treatment.It is assumed that starting from the microenvironment of cells in the early stage of carcinogenesis,regulating metabolic homeostasis,or targeting key genes can prevent disease progression at an early stage.Our previous study found that continuous induction of long-term low levels of IFN-γ can promote the malignant transformation of bovine mammary epithelial cells(BMECs).After 30 generations of IFN-γ treatment,BMECs(BMEC-30)showed enhanced proliferation and migration,accompanied by depletion of intracellular arginine and other characteristics similar to cancer cells.On this basis,this study,focusing on breast cancer,intends to clarify the role and mechanism of differential metabolites of mammary epithelial cells before and after malignant transformation on macrophage function from the perspective of metabolism,to provide the theoretical basis for understanding the influence of tumor microenvironment metabolite accumulation on immune function of the body.Methods:The present study is carried out by the bovine macrophage cell line(BOSMAC)as the object of investigation.IFN-γ induced supernatant of the 1st and 30 th generation BMECs culture was collected to simulate the microenvironment of normal cells and malignant cells.The effect of the microenvironment on macrophage function before and after malignant transformation was evaluated by CCK8,cytophagocytosis assay,and q RT-PCR.Targeted metabolomics was used to detect differential metabolites in the microenvironment before and after malignant transformation.CCK8,cytophagocytosis assay,q RT-PCR,flow cytometry,and Western blotting were used to determine the influence of differential metabolites on macrophage function.Construction(synthesis)and transfection of overexpressed plasmids(interfering plasmids)to verify key molecules of macrophage function regulated by differential metabolites by q RT-PCR and Western blotting;Immunohistochemistry and related databases were used to analyze the expression of key molecules in breast cancer samples and their relationship with the prognosis of breast cancer patients.Results:(1)Compared with normal cells,the culture supernatant of malignant epithelial cells inhibited the proliferation of macrophages,inhibited the overall level of macrophage phagocytosis of Staphylococcus aureus,inhibited the expressions of pro-inflammatory cytokines IL-1β,IL-6 and TNF-α,and enhanced the expression of anti-inflammatory cytokines IL-10.(2)Targeted metabolomics analysis showed that the contents of arginine and glutamine in the supernatant of malignant mammary epithelial cells were significantly decreased,while the contents of ornithine and citrulline were significantly increased.The depletion of arginine inhibited the proliferation of macrophages and the expression of pro-inflammatory factor IL-1β,IL-12 B and IL-6,and enhanced the phagocytosis activity of macrophages,the expression of antigen-presenting molecules MHC I and CD40,and the expression of anti-inflammatory factor IL-10.The addition of citrulline in the presence of glutamine significantly antagonized the effect of arginine depletion on the proliferation activity of macrophages.(3)Deficiency of arginine in the medium promotes the expression of activating factor 4(ATF4)of macrophages,Not only the inhibitory effect of arginine depletion on macrophage proliferation mediated by Cyclin A1,but also the anti-inflammatory ability of macrophages was enhanced.(4)Compared with paracancer tissue,the expression of ATF4 increased in breast cancer samples.With the progression of breast cancer,the high expression of ATF4 accompanied by decreased macrophage enrichment in the lesion is adverse to the recurrence-free survival(RFS)of patients.Conclusion:From the perspective of metabolism,this study elucidates the effect and mechanism of malignant mammaepithelial cells inhibiting macrophage immune activity by consuming large amounts of arginine,in which ATF4-mediated arginine depletion can inhibit macrophage proliferation and enhance anti-inflammatory ability.TISCH2 database shows that the expression of ATF4 in macrophages increases in breast cancer microenvironment.It deepens our understanding of the tissue microenvironment regulation of immune cell function,and provides targets and ideas for the early prevention and treatment of breast cancer and other cancers.