Immunosuppressive activity enhances central carbon metabolism and bioenergetics in myeloid-derived suppressor cells in vitro models

Abstract Introduction In some solid tumors it has been demonstrated that a subpopulation of myeloid cells, defined as “myeloid-derived suppressor cells” (MDSCs), plays an important role in inducing T cell tolerance by production of arginase 1 (arg1) that depletes microenvironment of arginine, an essential aminoacid for T cell function. Since chronic myeloid leukemia (CML) patients have high levels of immature myeloid cells it is of interest to investigate if these cells have MDSCs phenotype and activity. The aim of this study was to analyze MDSCs and investigate their activity in CML patients. Methods MDSCs were analyzed in peripheral blood (PB) of 20 healthy donors (HD) and 30 CML patients at diagnosis. In 21 patients MDSCs were also measured during TKI treatment. Granulocytic MDSCs (G-MDSCs) were identified as CD11b+CD33+CD14-HLADR- cells, while the monocytic MDSCs (Mo-MDSCs) as CD14+HLADR by cytofluorimetric analysis. Arg1 expression was assessed using real time PCR and Western Blot. Arg activity was measured in granulocyte lysates using a colorimetric test after enzymatic activation and arginine hydrolysis. Microvesicles (MV) were isolated from CML serum at diagnosis (n=5) by sequential ultracentrifugation. Results CML patients showed high levels of Mo- and G-MDSCs at diagnosis in comparison to HD (41±8 and 82,5±12,2% respectively for CML vs 9±2,1 and 55±5,3% for HD; p<0.001), while after TKIs therapy both subpopulations decreased, returning to normal values. T-reg (CD4+ CD25high Foxp3+ cells) were also significantly increased in CML patients at diagnosis in respect to HD (9±2% vs 6,1±0,8%, p<0.001) with a significant correlation with the percentage of Gr-MDSCs (r=0,6254; p<0.001). Both in PB and purified granulocytic cells, Arg1 expression showed a 30 fold increase in CML at diagnosis compared to HD (p<0.001) and decreased after therapy. The same data were confirmed by Western Blot analysis. Arg enzymatic activity in granulocytes resulted also increased in CML (n=10) compared to HD (n=10) (p<0.001). The suppressive function of CML G-MDSCs was demonstrated by their ability to inhibit the proliferation of CFSE+ HD T cells (p<0.001). In addition, an increase of Mo-MDSCs in vitro was observed after incubation of HD monocytes with CML sera (29±13%; p<0.0001) or MV (8±2,8%; p<0.05). Conclusions MDSCs are increased in CML patients at diagnosis and decrease during TKIs treatment. CML granulocytes have high arg1 activity and immunosuppressive activity. Moreover, CML serum as well as CML microvesicles increase the percentage of HD Mo-MDSCs. Disclosures: No relevant conflicts of interest to declare.

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Plant Mitochondrial Carriers: Molecular Gatekeepers That Help to Regulate Plant Central Carbon Metabolism

Plants ◽

10.3390/plants9010117 ◽

2020 ◽

Vol 9 (1) ◽

pp. 117 ◽

Cited By ~ 7

Author(s):

M. Toleco ◽

Thomas Naake ◽

Youjun Zhang ◽

Joshua Heazlewood ◽

Alisdair R. Fernie

Keyword(s):

Carbon Metabolism ◽

Tca Cycle ◽

Molecular Data ◽

Central Carbon Metabolism ◽

In Planta ◽

Inner Mitochondrial Membrane ◽

Mitochondrial Carriers ◽

Central Carbon ◽

Mitochondrial Carrier Family

The evolution of membrane-bound organelles among eukaryotes led to a highly compartmentalized metabolism. As a compartment of the central carbon metabolism, mitochondria must be connected to the cytosol by molecular gates that facilitate a myriad of cellular processes. Members of the mitochondrial carrier family function to mediate the transport of metabolites across the impermeable inner mitochondrial membrane and, thus, are potentially crucial for metabolic control and regulation. Here, we focus on members of this family that might impact intracellular central plant carbon metabolism. We summarize and review what is currently known about these transporters from in vitro transport assays and in planta physiological functions, whenever available. From the biochemical and molecular data, we hypothesize how these relevant transporters might play a role in the shuttling of organic acids in the various flux modes of the TCA cycle. Furthermore, we also review relevant mitochondrial carriers that may be vital in mitochondrial oxidative phosphorylation. Lastly, we survey novel experimental approaches that could possibly extend and/or complement the widely accepted proteoliposome reconstitution approach.

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Characterisation of putative lactate synthetic pathways of Coxiella burnetii

PLoS ONE ◽

10.1371/journal.pone.0255925 ◽

2021 ◽

Vol 16 (8) ◽

pp. e0255925

Author(s):

Janine Hofmann ◽

Mebratu A. Bitew ◽

Miku Kuba ◽

David P. De Souza ◽

Hayley J. Newton ◽

...

Keyword(s):

Enzyme Activity ◽

Coxiella Burnetii ◽

Carbon Metabolism ◽

Metabolic Pathways ◽

Malic Enzyme ◽

Lactate Production ◽

Central Carbon Metabolism ◽

Malolactic Enzyme ◽

Central Carbon

The zoonotic pathogen Coxiella burnetii, the causative agent of the human disease Q fever, is an ever-present danger to global public health. Investigating novel metabolic pathways necessary for C. burnetii to replicate within its unusual intracellular niche may identify new therapeutic targets. Recent studies employing stable isotope labelling established the ability of C. burnetii to synthesize lactate, despite the absence of an annotated synthetic pathway on its genome. A noncanonical lactate synthesis pathway could provide a novel anti-Coxiella target if it is essential for C. burnetii pathogenesis. In this study, two C. burnetii proteins, CBU1241 and CBU0823, were chosen for analysis based on their similarities to known lactate synthesizing enzymes. Recombinant GST-CBU1241, a putative malate dehydrogenase (MDH), did not produce measurable lactate in in vitro lactate dehydrogenase (LDH) activity assays and was confirmed to function as an MDH. Recombinant 6xHis-CBU0823, a putative NAD+-dependent malic enzyme, was shown to have both malic enzyme activity and MDH activity, however, did not produce measurable lactate in either LDH or malolactic enzyme activity assays in vitro. To examine potential lactate production by CBU0823 more directly, [13C]glucose labelling experiments compared label enrichment within metabolic pathways of a cbu0823 transposon mutant and the parent strain. No difference in lactate production was observed, but the loss of CBU0823 significantly reduced 13C-incorporation into glycolytic and TCA cycle intermediates. This disruption to central carbon metabolism did not have any apparent impact on intracellular replication within THP-1 cells. This research provides new information about the mechanism of lactate biosynthesis within C. burnetii, demonstrating that CBU1241 is not multifunctional, at least in vitro, and that CBU0823 also does not synthesize lactate. Although critical for normal central carbon metabolism of C. burnetii, loss of CBU0823 did not significantly impair replication of the bacterium inside cells.

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Implications of central carbon metabolism in SARS-CoV-2 replication and disease severity

10.1101/2021.02.24.432759 ◽

2021 ◽

Cited By ~ 1

Author(s):

Shuba Krishnan ◽

Hampus Nordqvist ◽

Anoop T. Ambikan ◽

Soham Gupta ◽

Maike Sperk ◽

...

Keyword(s):

Amino Acid ◽

Disease Severity ◽

Carbon Metabolism ◽

Metabolic Pathways ◽

Central Carbon Metabolism ◽

Lung Epithelial Cells ◽

Metabolic Adaptation ◽

Altered Expression ◽

Central Carbon

AbstractViruses hijack host metabolic pathways for their replicative advantage. Several observational trans-omics analyses associated carbon and amino acid metabolism in coronavirus disease 2019 (COVID-19) severity in patients but lacked mechanistic insights. In this study, using patient- derived multi-omics data and in vitro infection assays, we aimed to understand i) role of key metabolic pathways in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) reproduction and ii) its association with disease severity. Our data suggests that monocytes are key to the altered immune response during COVID-19. COVID-19 infection was associated with increased plasma glutamate levels, while glucose and mannose levels were determinants of the disease severity. Monocytes showed altered expression pattern of carbohydrate and amino acid transporters, GLUT1 and xCT respectively in severe COVID-19. Furthermore, lung epithelial cells (Calu-3) showed a strong acute metabolic adaptation following infection in vitro by modulating central carbon metabolism. We found that glycolysis and glutaminolysis are essential for virus replication and blocking these metabolic pathways caused significant reduction in virus production. Taken together, our study highlights that the virus utilizes and re-wires pathways governing central carbon metabolism leading to metabolic toxicity. Thus, the host metabolic perturbation could be an attractive strategy to limit the viral replication and disease severity.

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