Anne Claire Boschat (Presenter)
Institut Imagine
Bio: I studied at the European School of Biotechnology in Strasbourg (France). After obtaining both an french "diplôme d'Ingénieur" and a Master's degree in bioengineering, I worked for more than a year at New England Bioloabs in Boston, MA. I was then hired at the Institut Imagine (Paris, FR) to run the mass spectrometry platform that the institute shares with the Necker Hospital for Sick Children. The Institut Imagine then offered me the opportunity to join their fellowship program. I'm now a PhD student in addition to my work running the mass spectrometry platform.
Authorship: Anne-Claire Boschat (1,2,3), Norbert Minet (3,4) Emmanuel Martin (3,4), Robert Barouki (1,2,5), Sylvain Latour (3,4), Sylvia Sanquer (1,2)
(1) Plateforme de Métabolomique et Protéomique, Institut Imagine, Paris 75015, France (2) Laboratoire de Biochimie Métabolomique et Protéomique, Hôpital Necker Enfants-Malades, Paris 75015, France. (3) Université Paris Descartes Sorbonne Paris Cité, Institut Imagine, Paris 75015, France. (4) Laboratoire Activation Lymphocytaire et Susceptibilité à l’EBV, INSERM UMR 1163, Institut Imagine, Paris 75015, France. (5) INSERM UMR-S 1124, Université Paris Descartes, Centre Universitaire des Saints-Pères, Paris 75006, France.
| Short Abstract We recently identified a mutation in Cytosine 5’-Triphosphate Synthase 1 (CTPS1) leading to a severe immunodeficiency, characterized by an impaired capacity of activated T and B cells to proliferate. To better assess the role of CTPS in the onset of immunodeficiency, we first developed mass spectrometry targeted assays for measuring CTPS activity, nucleotides and deoxynucleotides. We are now developing large-scale metabolomics in order to provide usable data to further explore the altered metabolic pathways during the immune response and better characterize immune deficiencies. As a first attempt, we compared the metabolome of resting and stimulated lymphocytes. These first experiments validated our methodology and we are now in progress to perform studies on precious cells from PIDs patients. |
Long Abstract
Introduction
The human immune system is a complex mechanism protecting the organism against pathogens and infectious agents, such as viruses, bacteria, fungi, and other parasites, but that complexity makes it prone to malfunctions, amongst which: immunodeficiencies. There are over 100 recognized primary immunodeficiencies in human (PIDs) [1], most of which are very rare and due to genetic disorders. The majority are diagnosed in children under the age of one, although milder forms may not be recognized until adulthood. The intricacy of the network of regulations involved in controlling the immune system means a wide range of deregulated factors can induce PIDs, such as molecular defects in B and T cells, as is the case in nucleotides pool imbalance [2-5].
A severe metabolism adaptation is necessary in lymphocyte cell division after the antigen recognition takes place [6], and this adaptation is depending on DNA and RNA synthesis, and therefore of nucleic acids production. Nucleotides are the building blocks of nucleic acids, and enzymes involved in their synthesis, such as adenosine deaminase (ADA), purine nucleotide phosphorylase (PNP) and cytosine triphosphate synthase 1 (CTPS1) are essential for lymphocyte proliferation. Indeed, deficits in these enzymes are known to induce severe immunodeficiency [7-9].
Methods
In our laboratory, we have already undertaken the targeted detection of relevant markers of immune deficiencies such as nucleotides, deoxynucleotides and enzymatic activities of ADA, PNP and CTPS. We are now developing large-scale metabolomics in order to provide usable data to further explore the altered metabolic pathways during the immune response and better characterize immune deficiencies.
Metabolites were extracted from cell pellets according to the MPLEx protocol (Metabolite, Protein and Lipid extraction)) [10-11] using a mixture of methanol/water/chloroform (1/1/1). Lipids contained in the chloroform phase and proteins at the interphase were stored at -80°C to further lipidomics and proteomics analysis. The polar metabolites were analyzed by High Resolution Mass Spectrometry on a QExactive Plus system (Thermo) and run in positive and negative ESI modes. Two different chromatographic conditions based on Hydrophilic interaction (HILIC) and Reversed Phase C18 columns were used to analyze respectively polar and more hydrophobic metabolites. For the HILIC chromatographic conditions, the aqueous mobile phase solvent was 20 mM ammonium carbonate, adjusted to pH 9.4 with 0.1% ammonium hydroxide solution (25%) and the organic mobile phase was 100% acetonitrile. The polar metabolites were eluted from the column by using a linear gradient from 80% organic to 80% aqueous over 25 min. For the reversed phase C18 chromatographic conditions, the aqueous mobile phase solvent was 0.1% formic acid in water and the organic mobile phase was 0.1% formic acid in acetonitrile. The hydrophobic metabolites was eluted from the column by using a linear gradient from 100% aqueous to 90% organic over 25 min. The data analysis was performed on the XCMS online platform [12], created by the Scripps Research Institute (FL, USA), in three steps: data upload, parameters selection, and result interpretation with statistical analysis. Metabolomics features were defined as ions with a unique m/z and identification was done by matching the m/z measurements with the METLIN database.
Results
As a first attempt, we performed metabolomics studies on peripheral blood mononuclear cells (PBMC) stimulated or not with a mix of phorbol myristate acetate (PMA) and ionomycin to mimic lymphocyte growth stimulation. Results clearly show the well-known effect of lymphocyte stimulation, characterized by an increase of glycolysis metabolites along with nucleotides and amino acids production. An increase of lactate concentration was observed, concurrently with a decrease of metabolites involved in the tricarboxylic acid (TCA) cycle. Cell proliferation was more related to increase in aerobic glycolysis rather than oxidative phosphorylation [6]. The results will be presented during the congress. These first experiments validated our methodology and we are now in progress to perform studies on precious cells from PIDs patients.
Conclusions & Discussion
In conclusion, the goal of this ongoing study is to show the interest of metabolomics studies for comprehensive characterization of metabolites and metabolism in PID patients, helping to uncover the underlying causes of complex immune diseases.
References & Acknowledgements:
1. Picard C. et al. Primary Immunodeficiency Diseases: an Update on the Classification from the International Union of Immunological Societies Expert Committee for Primary Immunodeficiency 2015. J Clin Immunol. 35; 696–726 (2015)
2. Katherine M. Aird and Rugang Zhang Nucleotide Metabolism, Oncogene-Induced Senescence and Cancer. Cancer Lett. 28; 204–210 (2015)
3. Ammann AJ. Purine nucleotide imbalance in immunodeficiency disorders. Basic Life Sci. 31:487–502. (1985)
4. Boss GR, Seegmiller JE. Genetic defects in human purine and pyrimidine metabolism. Annu Rev Genet. 16;297–328 (1982)
5. Korte, D. et al. Imbalance in the Nucleotide Pools of Myeloid Leukemia Cells and HL-60 Cells: Correlation with Cell-Cycle Phase, Proliferation, Differentiation, and Transformation. Cancer Res. 47; 1841–1847 (1987).
6. MacIver, N. J., Michalek, R. D. & Rathmell, J. C. Metabolic Regulation of T Lymphocytes. Annu. Rev. Immunol. 31; 259–283 (2013).
7. Kathryn L. et al. Adenosine Deaminase (ADA)-Deficient Severe Combined Immune Deficiency (SCID): Molecular Pathogenesis and Clinical Manifestations. J Clin Immunol. 37; 626-637 (2017)
8. Somech, R. et al. Purine nucleoside phosphorylase deficiency presenting as severe combined immune deficiency. Immunol. Res. 56; 150-154 (2013)
9. Martin, E. et al. CTP synthase 1 deficiency in humans reveals its central role in lymphocyte proliferation. Nat. 510; 288–292 (2014).
10. Nakayasu, E. et al. MPLEx: a Robust and Universal Protocol for Single-Sample Integrative Proteomic, Metabolomic, and Lipidomic Analyses. mSystems. May-Jun; 1(3) (2016)
11. Contrepois, K. et al. Optimized Analytical Procedures for the Untargeted Metabolomic Profiling of Human Urine and Plasma by Combining Hydrophilic Interaction (HILIC) and Reverse-Phase Liquid Chromatography (RPLC)–Mass Spectrometry. Mol Cell Proteomics. 14; 1684-1695 (2015)
12. Huan, T. et al. Systems biology guided by XCMS Online metabolomics. Nat Meth. 14; 461-462 (2017)
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