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Immunology and Infection

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

Published: August 12th, 2016

DOI:

10.3791/54255

1Centre for Biomedical Research, Macfarlane Burnet Institute for Medical Research and Public Health, 2Department of Infectious Diseases, Monash University, 3Department of Microbiology and Immunology, University of Melbourne, 4Department of Microbiology, The University of the West Indies, 5Division of Experimental Medicine, Department of Medicine, University of California, San Francisco, 6Department of Medicine, Monash University

Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of monocytes induces effector functions and a concomitant shift from oxidative to glycolytic metabolism that is accompanied by increased glucose transporter expression. This increased glycolytic metabolism is also observed for trained immunity of monocytes, a form of innate immunological memory. Although in vitro protocols examining glucose transporter expression and glucose uptake by monocytes have been described, none have been examined by multi-parametric flow cytometry in whole blood. We describe a multi-parametric flow cytometric protocol for the measurement of fluorescent glucose analog 2-NBDG uptake in whole blood by total monocytes and the classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocyte subpopulations. This method can be used to examine glucose transporter expression and glucose uptake for total monocytes and monocyte subpopulations during homeostasis and inflammatory disease, and can be easily modified to examine glucose uptake for other leukocytes and leukocyte subpopulations within blood.

Monocytes are a major component of the human innate immune system that are rapidly mobilized to sites of infection and inflammation1. Activation of monocytes is critical for limiting acute damage by pathogens and is also central to the pathogenesis of several chronic diseases, including atherosclerosis2, cancer3, and HIV4,5.

The metabolism of resting and activated monocytes differs dramatically, with resting monocytes utilizing oxidative metabolism and activated monocytes utilizing glycolytic metabolism (i.e., fermentation of glucose to lactate)6. Activation of monocytes induce....

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NOTE: HIV-infected and HIV-uninfected subjects were recruited from the Infectious Diseases Unit at The Alfred Hospital in Melbourne, VIC, Australia, and from the local community, respectively. Informed consent was obtained from all participants, and the research was approved by The Alfred Hospital Research and Ethics Committee.

1. Glut1 Cell Surface Detection on Monocytes and Monocyte Subpopulations

  1. Collect blood in citrate ACD-B anticoagulant tubes and begin the experiments in a b.......

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Compensation must be performed for individual fluorochromes to prevent fluorescence spillover. Monocytes are first enriched by gating based on forward and side scatter. The plots presented are representatives of at least six independent experiments conducted on whole blood from six or more participants as previously reported10. Figure 1A shows the initial gating of monocytes by cell scatter and exclusion of T cells by gating within the CD3- populatio.......

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The protocol described here details a simple method to examine glucose transporter expression and fluorescent glucose analog uptake by monocyte and monocyte subpopulations in whole blood. By assessing 2-NBDG uptake in whole blood, this technique allows for conditions similar to those in vivo. A previous study examined 6-NBDG uptake in monocytes separated from whole blood by density centrifugation17. However, this study did not examine monocyte subpopulations and separation of monocytes from whole bloo.......

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This research was funded by the Australian Centre for HIV and Hepatitis Virology Research (ACH2) and a 2010 developmental grant (CNIHR) from the University of Washington Center for AIDS Research (CFAR), an NIH funded program under award number AI027757 which is supported by the following NIH Institutes and Centers (NIAID, NCI, NIMH, NIDA, NICHD, NHLBI, NIA). C.S.P is a recipient of the CNIHR and ACH2 grant. SMC is a recipient of a National Health and Medical Research Council of Australia (NHMRC) Principal Research Fellowship. The authors gratefully acknowledge the contribution to this work of the Victorian Operational Infrastructure Support Progr....

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Name Company Catalog Number Comments
VACUETT Tube 9 ml ACD-B anticoagulant tubes Greiner Bio-One GmbH 455094
5 ml sterile polypropylene tubes BD Biosciences 352063
Albumin from Bovine Serum (BSA) Sigma-Aldrich A7906
16% formaldehyde solution Electron Microscopy Science 15710
BD FACS lysing solution (10X) BD Biosciences 349202 Dilute BD FACS lysing solution 1/10 with deionized water for working concentration (store for up to 1 week at 4°C)
anti-CD3-PE BD Biosciences 555340
anti CD14-APC BD Biosciences 555399
anti-CD16-PECy7 BD Biosciences 557744
anti-Glut1-FITC R & D Systems FAB1418F
IgG2b-FITC R & D Systems IC0041F
2-NBDG Life technologies N13195 Suspend 5 mg of 2-NBDG into 1 ml of deionized water to make a 14.60 mM stock solution (keep for up to 6 months at 4°C). To make the working 2-NBDG concentration, dilute stock 1/100 with 1X DPBS. Cover with foil. (store for up to 1 week at 4°C)
Dulbecco’s Phosphate Buffered Saline (1X) Life technologies 14190-144 To make wash solution, add 0.5 g BSA per 100 ml DPBS (store for up to 2 weeks at 4°C)

  1. Shi, C., Pamer, E. G. Monocyte recruitment during infection and inflammation. Nat Rev Immunol. 11, 762-774 (2011).
  2. Woollard, K. J., Geissmann, F. Monocytes in atherosclerosis: subsets and functions. Nat Rev Cardiol. 7, 77-86 (2010).
  3. Richards, D. M., Hettinger, J., Feuerer, M. Monocytes and macrophages in cancer: development and functions. Cancer Microenviron. 6, 179-191 (2013).
  4. Anzinger, J. J., Butterfield, T. R., Angelovich, T. A., Crowe, S. M., Palmer, C. S. Monocytes as regulators of inflammation and HIV-related comorbidities during cART. J Immunol Res. 2014, 569819 (2014).
  5. Palmer, C., Cherry, C. L., Sada-Ovalle, I. Glucose Metabolism in T Cells and Monocytes: New Perspectives in HIV Pathogenesis. EBioMedicine. , (2016).
  6. Cheng, S. C., et al. mTOR- and HIF-1alpha-mediated aerobic glycolysis as metabolic basis for trained immunity. Science. 345, 1250684 (2014).
  7. Maratou, E., et al. Glucose transporter expression on the plasma membrane of resting and activated white blood cells. Eur J Clin Invest. 37, 282-290 (2007).
  8. Freemerman, A. J., et al. Metabolic reprogramming of macrophages: glucose transporter 1 (GLUT1)-mediated glucose metabolism drives a proinflammatory phenotype. J Biol Chem. 289, 7884-7896 (2014).
  9. Gonnella, R., et al. Kaposi sarcoma associated herpesvirus (KSHV) induces AKT hyperphosphorylation, bortezomib-resistance and GLUT-1 plasma membrane exposure in THP-1 monocytic cell line. J Exp Clin Cancer Res. 32, 79 (2013).
  10. Palmer, C. S., et al. Glucose transporter 1-expressing proinflammatory monocytes are elevated in combination antiretroviral therapy-treated and untreated HIV+ subjects. J Immunol. 193, 5595-5603 (2014).
  11. Wong, K. L., et al. Gene expression profiling reveals the defining features of the classical, intermediate, and nonclassical human monocyte subsets. Blood. 118, e16-e31 (2011).
  12. Ziegler-Heitbrock, L., et al. Nomenclature of monocytes and dendritic cells in blood. Blood. 116, e74-e80 (2010).
  13. Belge, K. U., et al. The proinflammatory CD14+CD16+DR++ monocytes are a major source of TNF. J Immunol. 168, 3536-3542 (2002).
  14. Frankenberger, M., Sternsdorf, T., Pechumer, H., Pforte, A., Ziegler-Heitbrock, H. W. Differential cytokine expression in human blood monocyte subpopulations: a polymerase chain reaction analysis. Blood. 87, 373-377 (1996).
  15. Ziegler-Heitbrock, L. The CD14+ CD16+ blood monocytes: their role in infection and inflammation. J Leukoc Biol. 81, 584-592 (2007).
  16. Ziegler-Heitbrock, L. . Macrophages: Biology and Role in the Pathology of Diseases. , 3-36 (2014).
  17. Dimitriadis, G., et al. Evaluation of glucose transport and its regulation by insulin in human monocytes using flow cytometry. Cytometry A. 64, 27-33 (2005).
  18. Fu, Y., Maianu, L., Melbert, B. R., Garvey, W. T. Facilitative glucose transporter gene expression in human lymphocytes, monocytes, and macrophages: a role for GLUT isoforms 1, 3, and 5 in the immune response and foam cell formation. Blood Cells Mol Dis. 32, 182-190 (2004).
  19. Stibenz, D., Buhrer, C. Down-regulation of L-selectin surface expression by various leukocyte isolation procedures. Scand J Immunol. 39, 59-63 (1994).
  20. Ahmed, N., Kansara, M., Berridge, M. V. Acute regulation of glucose transport in a monocyte-macrophage cell line: Glut-3 affinity for glucose is enhanced during the respiratory burst. Biochem J. 327 (Pt 2), 369-375 (1997).
  21. Cutfield, W. S., Luk, W., Skinner, S. J., Robinson, E. M. Impaired insulin-mediated glucose uptake in monocytes of short children with intrauterine growth retardation). Pediatr Diabetes. 1, 186-192 (2000).
  22. Yoshioka, K., et al. A novel fluorescent derivative of glucose applicable to the assessment of glucose uptake activity of Escherichia coli. Biochim Biophys Acta. 1289, 5-9 (1996).
  23. Speizer, L., Haugland, R., Kutchai, H. Asymmetric transport of a fluorescent glucose analogue by human erythrocytes. Biochim Biophys Acta. 815, 75-84 (1985).
  24. Palmer, C. S., et al. Increased glucose metabolic activity is associated with CD4+ T-cell activation and depletion during chronic HIV infection. AIDS. 28, 297-309 (2014).
  25. Palmer, C. S., Ostrowski, M., Balderson, B., Christian, N., Crowe, S. M. Glucose metabolism regulates T cell activation, differentiation, and functions. Frontiers in immunology. 6, (2015).
  26. Palmer, C. S., et al. Regulators of glucose metabolism in CD4 and CD8 T cells. International reviews of immunology. , 1-12 (2015).
  27. Palmer, C. S., Crowe, S. M. How does monocyte metabolism impact inflammation and aging during chronic HIV infection?. AIDS research and human retroviruses. 30, 335-336 (2014).
  28. McFadden, K., et al. Metabolic stress is a barrier to Epstein-Barr virus-mediated B-cell immortalization. Proceedings of the National Academy of Sciences of the United States of America. 113, E782-E790 (2016).
  29. Gamelli, R. L., Liu, H., He, L. K., Hofmann, C. A. Augmentations of glucose uptake and glucose transporter-1 in macrophages following thermal injury and sepsis in mice. Journal of leukocyte biology. 59, 639-647 (1996).
  30. Yin, Y., et al. Glucose Oxidation Is Critical for CD4+ T Cell Activation in a Mouse Model of Systemic Lupus Erythematosus. Journal of immunology. , 80-90 (2016).
  31. Yang, Z., Matteson, E. L., Goronzy, J. J., Weyand, C. M. T-cell metabolism in autoimmune disease. Arthritis research & therapy. 17, 29 (2015).
  32. Yin, Y., et al. Normalization of CD4+ T cell metabolism reverses lupus. Science translational medicine. 7, 274ra218 (2015).
  33. Barbera Betancourt, A., et al. Inhibition of Phosphoinositide 3-Kinase p110delta Does Not Affect T Cell Driven Development of Type 1 Diabetes Despite Significant Effects on Cytokine Production. PloS one. 11, e0146516 (2016).
  34. Barron, C. C., Bilan, P. J., Tsakiridis, T., Tsiani, E. Facilitative glucose transporters: Implications for cancer detection, prognosis and treatment. Metabolism: clinical and experimental. 65, 124-139 (2016).
  35. Hegedus, A., Kavanagh Williamson, M., Huthoff, H. HIV-1 pathogenicity and virion production are dependent on the metabolic phenotype of activated CD4+ T cells. Retrovirology. 11, 98 (2014).
  36. Taylor, H. E., et al. Phospholipase D1 Couples CD4+ T Cell Activation to c-Myc-Dependent Deoxyribonucleotide Pool Expansion and HIV-1 Replication. PLoS Pathog. 11, e1004864 (2015).
  37. Loisel-Meyer, S., et al. Glut1-mediated glucose transport regulates HIV infection. Proc Natl Acad Sci U S A. 109, 2549-2554 (2012).
  38. Palmer, C. S., et al. Emerging Role and Characterization of Immunometabolism: Relevance to HIV Pathogenesis, Serious Non-AIDS Events, and a Cure. J Immunol. 196 (11), 4437-4444 (2016).

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