Fig 2: xCT expression is increased in the CNS of rats with EAE. A. Histogram showing the neurological score during the course of acute EAE induced in Lewis rats by immunization with myelin basic protein. The peak of neurological disability was at day 14 post-immunization, which was selected for obtaining tissue samples. B. xCT mRNA (left) and protein (right) expression in spinal cord from control and acute EAE rats, as assessed by qPCR and Western blot analysis. Data are referred to mean expression level of controls (n = 5-6). C. Double immunofluorescence for xCT (green) and OX-42 (red), a marker of microglia and infiltrating macrophages. OX42+ cells express high xCT levels in acute EAE. Both meninges (asterisk in top) and infiltrating cells (bottom) in inflammatory foci show high levels of xCT in rat spinal cord with EAE as compared to controls. D. Microglial cells (OX42+ cells) of EAE rats have higher xCT levels in spinal cord than controls. Notice the difference between resting microglia in control rats, with ramified morphology (arrows in control) and microglia in EAE showing round shaped morphology, characteristic of its activated state (arrows in EAE). Scale bar = 20 µm.E. xCT mRNA (left) and protein (right) expression in spinal cord from control and chronic EAE mice, assessed by qPCR and Western blot analysis. Data are referred to mean expression level of controls (n = 5).

Fig 3: Activation of human peripheral blood monocytes by LPS induces glutamate release through the cystine/glutamate antiporter and xCT upregulation. A. Monocytes, isolated by MACS, showed an increase in glutamate release after their activation with LPS (1 µg/ml, 48 h). This effect is prevented in the presence of AAA (1 mM), an inhibitor of the cystine/glutamate antiporter. B. Flow cytometry analysis of peripheral blood monocytes (CD14-PE) reveals a change in the morphology of monocyte population after their activation with LPS (left-up). Staining with xCT-FITC demonstrates higher expression of this exchanger in LPS-activated CD14+ monocytes (left-down and right). *, p < 0.05; **, p < 0.01; #, p < 0.05 vs. LPS treatment.

Fig 4: xCT mRNA expression is increased in MS patients. A. Data show a significant increase in xCT mRNA expression in leukocytes in relapsing-MS, which is more prominent during relapses. Controls, n = 39; Total R-MS, n = 42; R-MS in remission, n = 24; R-MS in relapse, n = 18. B. Data show a significant increase in xCT mRNA in human optic nerve from MS patients as compared to matched controls. Damage optic nerves (DON), showing macroscopic plaques, atrophy and/or optic neuritis have a significantly higher increase in xCT mRNA. *, p < 0.05; **, p < 0.01; #, p < 0.05. C, D. xCT mRNA expression correlates with CD8 and EAAT2 mRNA expression in MS optic nerve. Pearson r = 0.83; n = 10; p = 0.0013 for CD8 vs. xCT. Pearson r = 0.81; n = 10; p = 0.005 for EAAT2 vs. xCT.

Fig 5: Activated U-937 monocytes release glutamate through cystine/glutamate antiporter and show an increased expression of the xCT subunit. A. Glutamate release by U-937 cells after activation with LPS (1 µg/ml) for 48 h in the absence and in the presence of the inhibitor of cystine/glutamate antiporter, AAA (1 mM), the glutaminase inhibitor DON (1 mM) and the inhibitors of glutamate transporters, DHK (1 mM), and TBOA (100 µM). Ordinates indicate the difference between the amount of glutamate released by LPS-activated and resting monocytes. Data are mean ± SEM from 4 independent experiments performed in triplicate. B, Intracellular glutathione levels in control U-937 cells and after activation with LPS (1 µg/ml) for 48 h. C. Relative expression of xCT antiporter and glutamate transporters EAAT1, EAAT2 and EAAT3 in CD14+ monocytes. D. Histogram illustrates the increase of xCT mRNA expression, but not of glutaminase (GLS), in LPS-activated U-937 monocytes using qPCR. U-937 cells were treated with LPS (1 µg/ml) for 48 h and qPCR data were normalized using 4 housekeeping genes and GeNorm software. Data are mean ± SEM from 3 independent experiments performed in triplicate. E. Western blotting analysis shows an up-regulation of xCT protein in U-937 cells after LPS (1 µg/ml) treatment for 48 ch. Data were normalized to actin and expressed as mean ± SEM from 3 independent experiments performed in triplicate. F. xCT mRNA levels in U-937 monocyte cell line increase after LPS (1 µg/ml) treatment but not after incubation with glutamate (100 µM and 1 mM) for 48 h. Stimulation with the cytokine TNFa (10 ng/ml; 24 h) also induced a significant increase in xCT mRNA expression. qPCR data were normalized using 4 housekeeping genes and GeNorm software. Data are mean ± SEM from 3 independent experiments performed in triplicate. *, p < 0.05; **, p < 0.01.