Metabolic response to glatiramer acetate therapy in multiple sclerosis patients

Abstract

Glatiramer acetate (GA; Copaxone) is a random copolymer of glutamic acid, lysine, alanine, and tyrosine used for the treatment of patients with multiple sclerosis (MS). Its mechanism of action has not been already fully elucidated, but it seems that GA has an immune-modulatory effect and neuro-protective properties. Lymphocyte mitochondrial dysfunction underlines the onset of several autoimmune disorders. In MS first diagnosis patients, CD4⁺, the main T cell subset involved in the pathogenesis of MS, undergo a metabolic reprogramming that consist in the up-regulation of glycolysis and in the down-regulation of oxidative phosphorylation. Currently, no works exist about CD4⁺ T cell metabolism in response to GA treatment. In order to provide novel insight into the potential use of GA in MS treatment, blood samples were collected from 20 healthy controls (HCs) and from 20 RR MS patients prior and every 6 months during the 12 months of GA administration. GA treated patients' CD4⁺ T cells were compared with those from HCs analysing their mitochondrial activity through polarographic and enzymatic methods in association with their antioxidant status, through the analysis of SOD, GPx and CAT activities. Altogether, our findings suggest that GA is able to reduce CD4⁺ T lymphocytes' dysfunctions by increasing mitochondrial activity and their response to oxidative stress.

Keywords: CAT, catalase; CD4+ T cells; CNS, central nervous system; CS, citrate synthase; EAE, experimental autoimmune encephalomyelitis; GPX, glutathione peroxidase; GR, glutathione reductase; Glycolysis; HK, hexokinase; MCT, mono-carboxylate transporters; MS, Multiple Sclerosis; Multiple sclerosis; OXPHOS; OXPHOS, oxidative phosphorylation; Oxidative stress; PBMC, peripheral blood mononuclear cell; PFK, phosphofructokinase; RCR, respiratory control ratio; ROS, reactive oxygen species; RRMS, Relapsing-Remitting Multiple Sclerosis; SOD, superoxide dismutase; Th, T helper.

Fig. 1

Respiratory rate of CD4 + T cells in controls and MS patients. Pyruvate/malate was used as respiratory substrate. Respiratory control rate [RCR] represents the ratio between state 3 and state 4 (table). Data are means ± S.E.M. from n = 20 controls and n = 20 MS subjects [0–6–12 months] each performed in triplicate. Asterisks indicate values that are significantly different from those obtained in control cells; ^⁎p < 0.05, ^⁎⁎p < 0.01, Student's t-test.

Fig. 2

Effect of Glatiramer acetate on the mitochondrial membrane potential of CD4⁺ T cells of controls and MS treated patients. JC-1 probe was used to assess variation of mitochondrial ΔΨ in controls [n = 20] and MS patients [n = 20]. Asterisks indicate values that are significantly different from those obtained in control cells; ^⁎p < 0.05, ^⁎⁎p < 0.01, Student's t-test.

Fig. 3

OXPHOS activity in CD4⁺ T cells. Citrate synthase-normalized activity of mitochondrial respiratory complex enzymes in CD4⁺ T cells. CD4⁺ T cells from control and MS treated subjects were assayed for enzymatic activities of electron transport chain complexes I-IV and citrate synthase. Figure depicts the relative activities compared to citrate synthase expressed as percent of the control group [A] and represent the means from n = 20 controls and n = 20 MS subjects [0–6–12 months of treatment], each performed in triplicate; bars indicate S.E.M. The same mitochondria-rich pellet utilized for OXPHOS activity determination was also used for CI-CIV expression: 15 μg were separated on a SDS-PAGE gel followed by western blot analysis using porin as a loading control. Representative immunoblot is shown [C]. Histograms show the mean values, expressed as percent of n = 10 controls and n = 10 MS subjects [B], each performed in triplicate; bars indicate S.E.M. Asterisks indicate values that are significantly different from those obtained in control cells; ^⁎p < 0.05, ^⁎⁎p < 0.01, Student's t-test.

Fig. 4

Activities of antioxidative enzymes: SOD, GPx and CAT. Antioxidant systems biomarkers in patients with MS and control subjects.[A] Superoxide dismutase [SOD], glutathione peroxidase [GPx] and catalase [CAT] activities were assayed in CD4⁺ T cells from control and MS treated subjects. Histograms show the mean values, expressed as percent of control, of n = 10 controls and n = 10 MS subjects, each performed in triplicate; bars indicate S.E.M. Asterisks denote values that are significantly different from those obtained in control cells; ^⁎p < 0.05, ^⁎⁎p < 0.01, Student's t-test.

Fig. 5

Bioenergetics of CD4 + T cells in control and MS subjects. CD4⁺ T cells from control and MS patients were assayed for [A] enzymatic glycolytic activities of hexokinase and phosphofructokinase, expressed as percent of control, [B] lactate production, [C] GLUT-1 and MCT-1 expression and for [D] ratio of mitochondrial over glycolytic ATP production (ratio Δ-lactate/basal lactate), where Δ-lactate is the difference of lactate production in presence and absence of antimycin A and basal lactate is lactate production in absence of antimycin. Cell lysates [30 μg] were separated on a SDS-PAGE gel followed by western blot analysis using β-actin as a loading control. Representative immunoblot is shown. Histograms show the mean values, expressed as percent of n = 10 controls and n = 10 MS subjects, each performed in triplicate; bars indicate S.E.M. Asterisks indicate values that are significantly different from those obtained in control cells; ^⁎p < 0.05, ^⁎⁎p < 0.01, Student's t-test.