Deletion of UCP2 in iNOS deficient mice reduces the severity of the disease during experimental autoimmune encephalomyelitis

Abstract

Uncoupling protein 2 is a member of the mitochondrial anion carrier family that is widely expressed in neurons and the immune cells of humans. Deletion of Ucp2 gene in mice pre-activates the immune system leading to higher resistance toward infection and to an increased susceptibility to develop chronic inflammatory diseases as previously exemplified with the Experimental Autoimmune Encephalomyelitis (EAE), a mouse model for multiple sclerosis. Given that oxidative stress is enhanced in Ucp2-/- mice and that nitric oxide (NO) also plays a critical function in redox balance and in chronic inflammation, we generated mice deficient for both Ucp2 and iNos genes and submitted them to EAE. Mice lacking iNos gene exhibited the highest clinical score (3.4+/-0.5 p<0.05). Surprisingly, mice deficient for both genes developed milder disease with reduced immune cell infiltration, cytokines and ROS production as compared to iNos-/- mice.

Figure 1. Score disease of mice upon EAE induction.

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 Methods). 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. *, P<0.05; **, P<0.01; ***, P<0.001.

Figure 2. Infiltration of immune cells in the central nervous system.

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 GADPH as the housekeeping gene. Statistical significance was determined by ANOVA. *, P<0.05; **, P<0.01; ***, P<0.001.

Figure 3. Relative expression of cytokines mRNA in CNS homogenates.

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 Materials and Methods. Data represent mean ± SEM of 7–12 mice per strain. *, P<0.05; **, P<0.01; ***, P<0.001.

Figure 4. ROS production in macrophages from Ucp2−/−, iNos−/−, and (Ucp2-iNos)−/− deficient mice.

Peritoneal macrophages were obtained at day 10 after MOG immunization and stimulated in vitro with 500 ng/ml phorbol merystil acetate during 1 hour as described in Materials and Methods. Data represent mean ± SEM from 5–13 mice per strain. Student's t test was performed to determine the statistical significance of the differences between Ucp2−/−, iNos−/− and (Ucp2-iNos)−/− mice. *, P<0.05; **, P<0.01.

Figure 5. Oxidized glutathione in the CNS.

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

Figure 6. The redox balance in macrophages during inflammation induced by EAE.

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).