Immunometabolic profiling of T cells from patients with relapsing-remitting multiple sclerosis reveals an impairment in glycolysis and mitochondrial respiration

Claudia La Rocca, Fortunata Carbone, Veronica De Rosa, Alessandra Colamatteo, Mario Galgani, Francesco Perna, Roberta Lanzillo, Vincenzo Brescia Morra, Giuseppe Orefice, Ilaria Cerillo, Ciro Florio, Giorgia Teresa Maniscalco, Marco Salvetti, Diego Centonze, Antonio Uccelli, Salvatore Longobardi, Andrea Visconti, Giuseppe Matarese, Claudia La Rocca, Fortunata Carbone, Veronica De Rosa, Alessandra Colamatteo, Mario Galgani, Francesco Perna, Roberta Lanzillo, Vincenzo Brescia Morra, Giuseppe Orefice, Ilaria Cerillo, Ciro Florio, Giorgia Teresa Maniscalco, Marco Salvetti, Diego Centonze, Antonio Uccelli, Salvatore Longobardi, Andrea Visconti, Giuseppe Matarese

Abstract

Background: Metabolic reprogramming is shaped to support specific cell functions since cellular metabolism controls the final outcome of immune response. Multiple sclerosis (MS) is an autoimmune disease resulting from loss of immune tolerance against central nervous system (CNS) myelin. Metabolic alterations of T cells occurring during MS are not yet well understood and their studies could have relevance in the comprehension of the pathogenetic events leading to loss of immune tolerance to self and to develop novel therapeutic strategies aimed at limiting MS progression.

Methods and results: In this report, we observed that extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), indicators of glycolysis and oxidative phosphorylation, respectively, were impaired during T cell activation in naïve-to-treatment relapsing remitting (RR)MS patients when compared with healthy controls. These results were also corroborated at biochemical level by a reduced expression of the glycolitic enzymes aldolase, enolase 1, hexokinase I, and by reduction of Krebs cycle enzymes dihydrolipoamide-S-acetyl transferase (DLAT) and dihydrolipoamide-S-succinyl transferase (DLST). Treatment of RRMS patients with interferon beta-1a (IFN beta-1a) was able to restore T cell glycolysis and mitochondrial respiration as well as the amount of the metabolic enzymes to a level comparable to that of healthy controls. These changes associated with an up-regulation of the glucose transporter-1 (GLUT-1), a key element in intracellular transport of glucose.

Conclusions: Our data suggest that T cells from RRMS patients display a reduced engagement of glycolysis and mitochondrial respiration, reversible upon IFN beta-1a treatment, thus suggesting an involvement of an altered metabolism in the pathogenesis of MS.

Keywords: Adipocytokines; IFN beta-1a; Metabolism; Multiple sclerosis.

Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Figures

Fig. 1
Fig. 1
Peripheral immunometabolic signature of RRMS patients before and after IFN beta-1a treatment. The graphs show the number per mm3 of several peripheral blood immune cell subpopulations and the serum concentration of several cytokines implicated in the control of metabolism and immune system functions in healthy controls (n = 24), naїve-to-treatment (n = 15) and IFN beta-1a treated (n = 15) RRMS patients. Data are shown as mean ± s.e.m. Comparisons were evaluated using non-parametric one-way ANOVA test (Kruskal-Wallis test) and Dunn's post-hoc test *P ≤ 0.05, **P ≤ 0.001, ***P ≤ 0.0001.
Fig. 2
Fig. 2
Impaired engagement of glycolysis and mitochondrial respiration of T cells from RRMS is reversed by IFN beta-1a treatment. The graphs represent the metabolic profile of PBMCs isolated from healthy controls, naїve-to-treatment and IFN beta-1a treated RRMS patients. (A) Kinetic profile of ECAR in PBMCs stimulated with OKT3 for 12 h. Healthy controls n = 25, naїve-to-treatment n = 11 and IFN beta-1a treated n = 17 RRMS patients. The data are shown as mean ± s.e.m. ECAR was measured in real time, under basal conditions and in response to glucose, oligomycin and 2-DG. Indices of glycolytic pathway activation, calculated from PBMCs ECAR profile: (B) basal, (C) maximal glycolysis and (D) glycolytic capacity. Data are expressed as mean ± s.e.m. (E) Kinetic profile of OCR in PBMCs stimulated with OKT3 for 12 h. Healthy controls n = 23, naїve-to-treatment n = 11 and IFN beta-1a treated n = 17 RRMS patients. The data are shown as mean ± s.e.m. OCR was measured in real time, under basal conditions and in response to oligomycin, FCCP, Antimycin A and Rotenone. Indices of mitochondrial respiratory function, calculated from PBMCs OCR profile: (F) basal, (G) maximal respiration and (H) spare capacity in PBMCs stimulated with OKT3 for 12 h. Data are expressed as mean ± s.e.m. Comparisons were evaluated using non-parametric one-way ANOVA test (Kruskal-Wallis test) and Dunn's post-hoc test *P ≤ 0.05, **P ≤ 0.001, ***P ≤ 0.0001.
Fig. 3
Fig. 3
Biochemical pathways of CD4+ T cells from RRMS patients before and after IFN beta-1a treatment. (A) Immunoblot for aldolase, hexokinase I, enolase 1, Glut-1, DLAT and DLST on CD4+ T cells from healthy control, naїve-to-treatment and IFN beta-1a treated RRMS patient upon 12 h anti-CD3/CD28 stimulation. Total ERK 1/2 served as a loading control. One representative out of at least three independent experiments. (B) The graphs show the relative densitometric quantitation of aldolase, hexokinase I, enolase 1, Glut-1, DLAT and DLST normalized on total ERK1/2 in healthy controls, naїve -to-treatment and IFN beta-1a treated RRMS patients. One representative experiment is shown, from one individual for each condition; in total we runned al least 3 healthy controls, 3 naive-to-treatment and 3 RRMS IFN beta-1a treated patients. We scanned 3 times at least three films with different exposures from each subject, and averaged values were utilized as densitometry to reduce variations among samples. Comparisons were evaluated using non-parametric one-way ANOVA test (Kruskal-Wallis test) and Dunn's post-hoc test *P ≤ 0.05, **P ≤ 0.001, ***P ≤ 0.0001.

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