Project description:To recruit phagocytes, apoptotic cells characteristically release ATP, which functions as a “danger” signal. Here, we found that the culture supernatant of apoptotic cells activated the macrophages to express anti-inflammatory genes such as NR4A and Thbs1. A high level of AMP accumulated in the apoptotic cell supernatant in a Pannexin1-dependent manner. A nucleotidase inhibitor and A2a adenosine receptor antagonist inhibited the apoptotic supernatant-induced gene expression, suggesting AMP was metabolized to adenosine by an ecto-5’-nucleotidase expressed on macrophages, to activate the macrophage A2a adenosine receptor. Intraperitoneal injection of zymosan into AdoR A2a- or Panx1-deficient mice produced high, sustained levels of inflammatory mediators in the peritoneal lavage. These results indicated that AMP from apoptotic cells suppresses inflammation as a “calm down” signal. If apoptotic cells produce “danger” or “anti-danger” signal(s), we rationalized that such signals would activate gene expression in macrophages. To investigate this possibility, we examined the effect of the culture supernatant from apoptotic cells on macrophage gene expression by using microarrays. For mouse BMDMs, bone marrow cells from female C57BL/6J mice at 8 weeks of age were cultured for more than 7 days with DMEM containing 10% FCS supplemented with mouse M-CSF. We used adherent cells as BMDMs in the study. W3 cells, mouse T cell line expressing Fas, were treated with human Fas ligand at 37°C for 30 min to induce apoptosis. The cells were then washed and re-suspended at a concentration of 1 × 107 cells/ml with RPMI containing 1% FCS, and further incubated for 60 min at 37°C. Following Fas ligand treatment, more than 90% of the W3 cells were Annexin V positive, and only small percentage were positive for both Annexin V and propidium iodide (PI). The culture supernatant was collected from apoptotic W3 cells. Next, BMDMs were incubated with medium (BMDMs-Medium) or apoptotic W3 cell supernatant (BMDMs-Apoptotic cell supernatant) for 1 h. Total RNA was extracted from the cells and hybridized on Affymetrix microarrays.

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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Expression data from mouse rGAS6-stimulated BMDMs

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