Construction Of Macrophage Lines With Stable Knockout Of MCART-1 Gene And The Studies Of Their Inflammatory Phenotypic

Background and Objects:Macrophages play an important role in tumor growth,inflammatory response,and wound repair.Macrophages are a functionally heterogeneous cell population that is mainly shaped by a variety of microenvironmental stimuli.In vitro,Interferon y(IFN-y),interleukin-1β(IL-1β),and lipopolysaccharide(LPS)induce a classical activation of macrophages(M1),whereas IL-4 and IL-13 induce an alternative activation program in macrophages(M2).M1 is involved in the development of acute inflammation,and the alternatively activated M2 phenotype plays an important role in the process of inflammation resolution and wound repair[1].Reprogramming of intracellular metabolisms is required for the proper polarization and functions of activated macrophages[2].M1 macrophages increase glucose consumption and lactate release and decreased oxygen consumption rate.In comparison,M2 macrophages mainly employ oxidative glucose metabolism pathways.Mitochondrial metabolites and their derivatives also have important inflammatory modulatory effects,such as acetyl-CoA,succinic acid,α-ketoglutaric acid,itaconic acid and reactive oxygen species,which can regulate the expression of M1-type pro-inflammatory mediators by influencing epigenetic regulation and activating hypoxia-inducible factor-1 α(HIF-1α)and IκB kinase(IKK)[3-6].In M2 macrophages,mitochondrial oxidative respiration-produced intermediate metabolites and energy are involved in maintaining the proliferation and anti-inflammatory phenotype of M2 macrophages[6-8].Thus,interference with mitochondrial metabolism may significantly affect the inflammatory phenotype and immune function of macrophages.Mitochondrial Nicotinamide adenine dinucleotide(NAD+)participates in the whole process of tricarboxylic acid cycle and oxidative phosphorylation as an electron acceptor,and interference with mitochondrial NAD levels can affect the levels of several mitochondrial metabolites and ATP.In 2020,the mammalian mitochondrial NAD+transporter MCART1/SLC25A51 was first identified[9].Knocking out the MCART-lgene significantly reduces the transport of NAD+in the cytoplasm to mitochondria,reducing the levels of several mitochondrial metabolites and ATP[9,10].Since mitochondrial energy metabolism and substance metabolism play an important role in regulating the inflammatory phenotype of macrophages,we suspect that interference with MCART-1 gene expression may affect the proliferation and inflammatory phenotype of macrophages and may have potential therapeutic effects in the treatment of inflammatory diseases and malignancies involving macrophages.In this project,we use CRISPR/Cas9 to knock out the MCART-1 gene in macrophages,and the RAW264.7 cell line with the MCART-1 gene stably knocked out was constructed,and the proliferation and energy metabolism changes of this cell line were preliminarily explored,and the expression changes of several cancer-promoting mediators under the induction conditions of glioma GL261were preliminarily explored.It provides an effective tool for further study of the role of MCART-1 gene in regulating the mitochondrial metabolism and macrophages inflammatory phenotype.MethodsIn vitro,wild type RAW264.7 cells(WT)were infected with Cas9 lentiviral vector and sgRNA lentiviral vector,respectively,and RAW264.7 cell lines(RAW264.7-cas9)with stable Cas9 protein expression and macrophage RAW264.7 cell lines(KO)with MCART-1 gene stably knocked out were constructed successively;by single-cell sequencing,PCR and Western blot et al.identified the knockout effect of MCART-1 gene;Seahorse,transcriptomics,metabolomics and other methods were used to study the effect of knocking out the MCART-1 gene on energy metabolism and material metabolism of macrophages.The effect of knocking out MCART-1 gene on macrophage proliferation was studied by CFSE and cell counting.GL261 tumor-conditioned medium was used to induce cell polarization into tumor-associated macrophages,and the effect of knocking out MCART-1 gene on the inflammatory phenotype and immune function of tumor-associated macrophages was studied by real-time PCR experiments and flow cytometry.Results1.Successful construction of macrophage RAW264.7 with MCART-1 gene stably knocked out.2.Knocking out the MCART-1 gene reduced the rate of oxidative phosphorylation of RAW264.7 cells and increased the rate of glycolysis.3.Knocking out of the MCART-1 gene reduced mitochondrial ATP production in RAW264.7 cells and increases glycolytic ATP production.4.Knocking out of the MCART-1 gene reduced aspartic acid biosynthesis,nucleotide levels,and the proliferation rate of RAW264.7 cells.5.Knocking out of the MCART-1 gene decreased the expression of SIRPa in tumorassociated macrophages and enhances the phagocytic function of tumor-associated macrophages.6.Knocking out of the MCART-1 gene reduced the expression of HIF-1α、inflammatory factors IL-1β and IL-6 and cancer-promoting mediators TGFβ and IL-10 in tumorassociated macrophage.7.Knocking out of the MCART-1 gene altered the whole gene transcription level of macrophage.Conclusion:Knocking out of the MCART-1 gene reduced the proliferation and oxidative phosphorylation of RAW264.7,downregulated the expression of inflammatory factors and immunosuppressive factors,and may improve the inflammatory phenotype and immunosuppressive microenvironment of glioma-associated macrophages.