Catala Alexis, Youssef Lyla A, Reisz Julie A, Dzieciatkowska Monika, Powers Nicholas E, Marchetti Carlo, Karafin Matthew, Zimring James C, Hudson Krystalyn E, Hansen Kirk C, Spitalnik Steven L, D'Alessandro Angelo
Department of Biochemistry and Molecular Genetics, University of Colorado Denver - Anschutz Medical Campus, Aurora, CO, United States.
Program in Structural Biology and Biochemistry, University of Colorado Denver - Anschutz Medical Campus, Aurora, CO, United States.
Front Physiol. 2020 Apr 30;11:396. doi: 10.3389/fphys.2020.00396. eCollection 2020.
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 mouse 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-C 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 C-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.
网状内皮巨噬细胞每天通过一种称为红细胞吞噬作用（EP）的过程吞噬约2万亿个衰老红细胞。这一关键机制通过调节红细胞（RBC）分解代谢和铁循环来维持全身血红素铁稳态。尽管大量研究表明促炎细胞因子刺激对巨噬细胞代谢重编程有多种影响，但关于EP对巨噬细胞代谢组和蛋白质组的影响知之甚少。因此，我们对IgG包被的红细胞进行EP前后的小鼠骨髓来源巨噬细胞（BMDM）进行了基于质谱的代谢组学和蛋白质组学分析。此外，对与游离IgG孵育的BMDM进行了代谢组学分析，以确保巨噬细胞代谢的变化是由于调理素化红细胞而非游离IgG结合所致。在有和没有IgG包被红细胞的情况下，对BMDM进行了均匀标记示踪实验，以评估葡萄糖通过磷酸戊糖途径（PPP）的通量。在本研究中，我们证明EP显著改变氨基酸和脂肪酸代谢、三羧酸循环、氧化磷酸化和花生四烯酸-亚油酸代谢。EP后BMDM中氨基酸、脂质和氧化脂质、血红素产物以及RBC衍生蛋白水平升高。用U-C葡萄糖进行的示踪实验表明糖酵解通量减慢，PPP激活增强。值得注意的是，我们发现它由EP前巨噬细胞自身或细胞外培养基中的葡萄糖提供能量，而非来自调理素化红细胞。PPP衍生的NADPH随后可为氧化爆发提供能量，导致通过自由基攻击促进吞噬的RBC蛋白消化所需的活性氧生成。氧化还原蛋白质组学实验证实了结果，分别证明了甘油醛-3-磷酸脱氢酶（GAPDH）的Cys152和血红蛋白-β的Cys94被氧化。三羧酸循环早期和C-分支二元酸代谢物（分别为α-酮戊二酸和2-羟基戊二酸）的显著增加表明EP促进线粒体代谢失调。最后，IgG介导的RBC摄取后，ALA和鸟氨酸的显著积累表明EP刺激了δ-氨基-γ-酮戊酸（ALA）合酶和精氨酸酶活性。重要的是，单独暴露于IgG后不会发生EP介导的BMDM代谢重编程。总之，我们表明EP对巨噬细胞代谢进行重编程并改变巨噬细胞极化。
