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Next Generation Sequencing Analysis of WT and Hectd3–/– BMDMs during F. novicida infection

ABSTRACT: Purpose: The goals of this study are to compare NGS-derived WT and Hectd3–/– transcrip profiling (RNA-seq) in the absence and presence of F. novicida infection Methods: Bone marrow derived macrophages (BMDMs) from WT and Hectd3–/– mice were infected with F .novicida for 8 and 12 hours. mRNA profiles were generated by deep sequencing using Illumina GAIIx. Results: Using an optimized data analysis workflow, we mapped about 80 million sequence reads per sample (around 95% clean reads were successfully mapped) to the mouse genome and identified 22,589 transcripts in the WT and Hectd3–/– BMDMs with or without F. novicida infection. Conclusions: Our study represents the first detailed analysis of HECTD3 deficiency regulated gene expression in response to F. novicida infection generated by RNA-seq technology. Our results demonstrated that the type I IFN signaling induced by F. novicida infection was impaired in the absence of HECTD3.

ORGANISM(S): Mus musculus

PROVIDER: GSE104127 | GEO | 2018/06/15

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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Next Generation Sequencing Analysis of WT and Hectd3–/– BMDMs during F. novicida infection

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