Multi-omics Analysis Identifies FoxO1 As A Regulator Of Macrophage Function Through Metabolic Reprogramming

Macrophages are plastic cells that can switch among different states according to bioenergetic or biosynthetic requirements.Transcription factor Forkhead Box Protein 1(FoxO1)is an important member of the FoxO family.Our previous work demonstrated that FoxO1 plays a pivotal role in regulating the function of tumor associated macrophages.FoxO1 deficient tumor associated macrophages promote tumor progression,but the underlying mechanisms are still unclear.In this study,we mainly focused on the perspective of the transcription factor FoxO1 participating in metabolic regulation,and exploring the molecular mechanism that affects the function of macrophages.The main experimental results obtained are as follows:1.FoxO1mKO macrophages are skewed toward a M2 Phenotype in Physiological State.First,we constructed mice with FoxO1 specific deficient macrophages,and used bone marrow-derived macrophages(BMDMs)for transcriptome and proteome analysis.The results show that FoxO1 deficiency results in an M2 phenotype of macrophages.Furthermore,we used high content screening and analysis technology system to further confirm that phagocytosis function of FoxO1-deficient macrophages on B16-F10mcherry tumor cells decreased significantly,verifying that FoxO1 can regulate the phenotype and function of macrophages.2.Glycolytic activity is downregulated in FoxO1-deficient macrophages.Next,we explore the detailed molecular mechanism of the transcription factor FoxO1 on regulating macrophages.Our GSEA and heatmap showed that the genes associated with Reactome glycolysis reduced in FoxO1mKO macrophages compared with those of FoxO1fl/fl macrophages in physiological state according to transcriptome and proteomics data,suggesting that FoxO1 plays an important role in the process of macrophage metabolism.Furthermore,we used the shRNA interference technology and flow cytometry technology to confirm that FoxO1 regulating the expression of key glycolysis gene(PKM2,LDHA,etc.),and the glucose intake is significantly reduced in macrophages lacking FoxO1.These suggest that FoxO1 affects the function of macrophages by regulating metabolic reprogramming.3.FoxO1 regulates glucose metabolism of macrophages to affect macrophages function.To further confirm the role of FoxO1 in regulating glucose metabolism in macrophages,we measured the kinetic extracellular acidification rate(ECAR)response curve in BMDMs from FoxO1-deficient mice and littermate controls,and we found that the ECAR was lower in FoxO1-deficient macrophages than in normal macrophages.In addition,after pre-treatment with the glycolysis inhibitor 2-DG,FoxO1-deficient macrophages showed a higher cell count than WT macrophages when co-cultured with B16-F10mCherry cells.FoxO1-siRNA knockout macrophages further confirmed that FoxO1 participates in the process of glycolysis metabolism and then regulates the function of macrophages.In summary,we found that the transcription factor FoxO1 regulating the function of macrophages by participating in metabolic reprogramming,and FoxO1 serves as a bridge between metabolism and macrophage function.