ROS production in macrophages from <i>Ucp2−/−</i>, <i>iNos−/−</i>, and <i>(Ucp2-iNos)−/−</i> deficient mice.

<p>Peritoneal macrophages were obtained at day 10 after MOG immunization and stimulated <i>in vitro</i> with 500 ng/ml phorbol merystil acetate during 1 hour as described in <i>Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022841#s4" target="_blank">Methods</a></i>. Data represent mean ± SEM from 5–13 mice per strain. Student's <i>t</i> test was performed to determine the statistical significance of the differences between <i>Ucp2−/−</i>, <i>iNos−/−</i> and <i>(Ucp2-iNos)−/−</i> mice. *, <i>P</i><0.05; **, <i>P</i><0.01.</p

Infiltration of immune cells in the central nervous system.

<p>Both brain and spinal cord from MOG immunized mice of all genotypes were collected at days 10 (white) and day 14 (grey). Levels of mRNAs specific for the monocyte cell marker CD11b (A), and for the T cell markers CD8 (B) and CD4 (C) were assessed by real time quantitative RT-PCR. The data are expressed as the relative mean ± SEM of 9–12 mice per genotype using <i>GADPH</i> as the housekeeping gene. Statistical significance was determined by ANOVA. *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

The redox balance in macrophages during inflammation induced by EAE.

<p>Once activated, Th1 cells enter the CNS and produce proinflamatory cytokines (IFNγ, Il2) that stimulate resident macrophages. Inflammation is further increased with the autocrine production of TNFα by macrophages. At the cellular level, ROS production stimulates the expression of proinflammatory genes such as the NO synthase gene. In one hand, nitric oxide inhibits the respiratory chain (RC), which, in turn, increases the mitochondrial ROS production and consequently oxidative cell damage. In the other hand, NO prevent Th1 cell expansion and inhibits the NAPH oxidase enzyme, an essential ROS producer that trigger tissue damage and brain blood barrier destruction. At the molecular level, NADPH plays a pivotal function in the redox balance as substrate of the NADPH oxidase, the NO sytnhase (iNOS) and the gluthation reductase (GR). In this context, mitochondrial carriers, including UCP2, could modulate the availability of this substrate. For instance the citrate carrier supplies an additional source of cytosolic NADPH by exporting citrate that is further oxidized into alpha keto glutarat (α-KG) by the cytosolic isocitrate dehydrogenase (ICDH).</p

Score disease of mice upon EAE induction.

<p>EAE was induced in 7–10 weeks of age mice by subcutaneous injection with 150 µg MOG_35–55 peptide in presence of Freund adjuvant and Pertussis toxin (see Material and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022841#s4" target="_blank">Methods</a>). Clinical symptoms were scored as follows: 0, normal; 1, weak/flaccid tail; 2, waddle; 3, moderate paraparesis; 4, severe paraparesis; 5, tetraparesis; 6, moribund. The mean clinical score was determined as the average score of all animals for a given genotype at days 10, 13, 14 and 15. Statistical significance was determined by one way ANOVA and free parametric Kruskal-Wallis test. *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

Oxidized glutathione in the CNS.

<p>Oxidized glutathione was assayed 14 days after EAE induction, as described in experimental procedures, in the CNS from <i>Ucp2−/−</i>, <i>iNos−/−</i> and <i>(Ucp2-iNos)−/−</i> deficient mice. Data represents means ± SEM of 4–5 mice per genotype.</p

Relative expression of cytokines mRNA in CNS homogenates.

<p>Fourteen days after EAE induction, brains and spinal cords were collected and levels of mRNAs were assessed by real time quantitative PCR as described in <i>Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022841#s4" target="_blank">Methods</a></i>. Data represent mean ± SEM of 7–12 mice per strain. *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

Antioxidant enzymatic activities.

<p>Comparison between <i>Cftr</i>^+/+ and <i>Cftr</i>^−/− mice of SOD (A), GPx (B) and CAT (C) activities in lung homogenates. Enzymatic activities were measured by standard spectrophotometric methods. Lung homogenates from <i>Cftr</i>^−/− mice displayed significantly higher SOD and GPx activities than those from <i>Cftr</i>^+/+ mice. Both <i>Cftr</i>^+/+ and <i>Cftr</i>^−/− mice displayed a similar level of CAT activity. Values are presented as specific activity (mean±S.E., n = 5, in duplicate). * P≤0.05</p

Phosphatidylcholine hydroperoxides quantification.

<p><i>Cftr</i>^+/+ and <i>Cftr</i>^−/− mice were injected I.P. twice with paraquat (PQ) (+) or saline (−) at 24 h intervals and sacrificed 48 h after the first injection. After lipid extraction, phosphatidylcholines (PC) and their corresponding hydroperoxides (PC-OOH) were separated by HPLC and detected by UV and chemiluminescence. After PQ challenge PLPC-OOH (16∶0–18∶2), PAPC–OOH (16∶0–20∶4) and PDPC–OOH (16∶0–22∶6) were significantly higher in <i>Cftr</i>^−/− lung compared to <i>Cftr</i>^+/+. Data are expressed as the ratio of PC–OOH to PC peak heights (mean±S.E.) and are representative of 3 different experiments with at least n = 3 animals in each experiment. * P≤0.05, *** P≤0.001</p

Prdx6 protein and mRNA levels after paraquat exposure.

<p><i>A) Prdx6 protein expression levels after paraquat (PQ) treatment</i>. Lung protein extracts from <i>Cftr</i>^+/+ and <i>Cftr</i>^−/− mice treated with either PQ (+) or saline (−) were subjected to immunoblot analysis using antibodies against Prdx6. The decrease in Prdx6 protein after PQ exposure is greater in <i>Cftr</i>^−/− mice than in <i>Cftr</i>^+/+. On the left, a representative blot is presented. Individual data were quantified as ratio of fluorescence intensity for Prdx6 bands to the intensity obtained for α-tubulin. Data are shown as percent of <i>Cftr</i>^+/+ treated with saline (mean±S.E., n = 5 for saline and n = 10 for PQ). <i>B) Prdx6 mRNA levels after PQ treatment</i>. Total lung RNA from <i>Cftr</i>^+/+ and <i>Cftr</i>^−/− mice treated with either PQ (+) or saline (−) was reversely transcribed and the amount of cDNAs coding for Prdx6 and the reference protein β-actin were measured by quantitative Real-Time PCR. The decrease in Prdx6 mRNA after PQ challenge is greater in <i>Cftr</i>^−/− mice than in <i>Cftr</i>^+/+. Indicated values correspond to the ratio of Prdx6 to β-actin mRNA (mean±S.E., n = 8 for saline and n = 5 for PQ, in triplicate). <i>C) Immunohistochemical analysis of Prdx6 after PQ</i> treatment. Acetone-fixed cryosections from <i>Cftr</i>^+/+ and <i>Cftr</i>^−/− mice treated with PQ or saline were incubated with anti-Prdx6 antibodies and visualized by confocal microscopy. Panels correspond to sections of bronchioles from <i>Cftr</i>^+/+ mice treated with either saline (a) or PQ (b) and from <i>Cftr</i>^−/− mice treated with saline (c) or PQ (d). Prdx6 staining intensity was lower after PQ exposure. * P≤0.05, *** P≤0.001</p

Cysteine 47 overoxidation to sulfonic acid in Prdx6 extracted from pI 6.3 spot.

<p>Fragmentation spectra of DFTPVC(SO3H)TTELGR peptide, corresponding to Prdx6 amino acid residues 42–53, unequivocally indicates oxydation of Cys47 to sulfonic acid. The most abundant peaks (singly charged) of the peptide are annotated as b- and y- series daughter ions, in red and blue respectively. Fragmentation spectra of the non-oxidized peptide show a similar fragmentation pattern, with a mass shift of 48 Da on peaks with oxidized cysteine (data not shown).</p