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Gene expression analysis of effects of Met on BMDMs co-cultured with COM-TECs.

ABSTRACT: Microarray analysis was used to assess the expression levels of mRNAs in bone marrow-derived macrophages (BMDMs) pretreated with metformin (Met) or PBS and co-cultured with renal tubular epithelial cells (TECs) or COM-TECs (NC vs. COM, COM vs. COM + Met).BMDMs and TECs were isolated from wild-type (WT) C57BL/6J mice. To developed a BMDM-COM-stimulated TECs co-culture system, BMDMs were plated in the upper chamber of 6-well Transwell plates with a pore size of 0.4 μm (Corning, USA), while TECs were plated in the lower chamber. In the COM and COM + Met groups, TECs were treated with COM (100 μg/mL) for 24 h and BMDMs were treated with Met (5.0mM) for 24 h.

ORGANISM(S): Mus musculus

PROVIDER: GSE192593 | GEO | 2021/12/29

REPOSITORIES: GEO

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Project description:Reticuloendothelial macrophages engulf ~0.2 trillion senescent erythrocytes daily in a process called erythrophagocytosis (EP). This critical mechanism preserves systemic heme-iron homeostasis by regulating red blood cell (RBC) catabolism and iron recycling. Although extensive work has demonstrated the various effects on macrophage metabolic reprogramming by stimulation with proinflammatory cytokines, little is known about the impact of EP on the macrophage metabolome and proteome. Thus, we performed mass spectrometry-based metabolomics and proteomics analyses of bone marrow-derived macrophages (BMDMs) before and after EP of IgG-coated RBCs. Further, metabolomics was performed on BMDMs incubated with free IgG to ensure that changes to macrophage metabolism were due to opsonized RBCs and not to free IgG binding. Uniformly labeled tracing experiments were conducted on BMDMs in the presence and absence of IgG-coated RBCs to assess the flux of glucose through the pentose phosphate pathway (PPP). In this study, we demonstrate that EP significantly alters amino acid and fatty acid metabolism, the Krebs cycle, OXPHOS, and arachidonate-linoleate metabolism. Increases in levels of amino acids, lipids and oxylipins, heme products, and RBC-derived proteins are noted in BMDMs following EP. Tracing experiments with U-13C6 glucose indicated a slower flux through glycolysis and enhanced PPP activation. Notably, we show that it is fueled by glucose derived from the macrophages themselves or from the extracellular media prior to EP, but not from opsonized RBCs. The PPP-derived NADPH can then fuel the oxidative burst, leading to the generation of reactive oxygen species necessary to promote digestion of phagocytosed RBC proteins via radical attack. Results were confirmed by redox proteomics experiments, demonstrating the oxidation of Cys152 and Cys94 of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and hemoglobin-β, respectively. Significant increases in early Krebs cycle and C5-branched dibasic acid metabolites (α-ketoglutarate and 2-hydroxyglutarate, respectively) indicate that EP promotes the dysregulation of mitochondrial metabolism. Lastly, EP stimulated aminolevulinic acid (ALA) synthase and arginase activity as indicated by significant accumulations of ALA and ornithine after IgG-mediated RBC ingestion. Importantly, EP-mediated metabolic reprogramming of BMDMs does not occur following exposure to IgG alone. In conclusion, we show that EP reprograms macrophage metabolism and modifies macrophage polarization.

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Gene expression analysis of effects of Met on BMDMs co-cultured with COM-TECs.

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