Uncoupling protein 2 has protective function during experimental autoimmune encephalomyelitis

Abstract

Uncoupling protein 2 (UCP2) is a member of the mitochondrial transporter superfamily that is expressed in many tissues, including immune cells. UCP2 prevents oxidative stress by reducing reactive oxygen species. Using UCP2-deficient mice, it was shown that UCP2 is involved in the regulation of insulin secretion, in the resistance to infection, and in atherosclerosis. Here, we investigated the role of UCP2 in experimental autoimmune encephalomyelitis, a murine model of multiple sclerosis. Immunized C57BL/6J UCP2-deficient mice showed a slightly delayed onset during experimental autoimmune encephalomyelitis (13.0 +/- 0.6 versus 11.5 +/- 0.8 in wild-type controls) and developed significantly higher disease scores than littermate controls (maximum disease score of 2.9 +/- 0.2 versus 1.7 +/- 0.2, P = 0.001). Higher levels of infiltrating T cells into the spinal cord meninges and parenchyma were observed. The T-cell proliferative response to the specific antigen was increased in UCP2-deficient mice compared with littermate controls, and CD4 cells of UCP2 knockout mice produced significantly higher levels of pro-inflammatory cytokines, eg, tumor necrosis factor-alpha and interleukin-2, resulting from a Th1 response. Mice lacking UCP2 also developed a higher B-cell response. Concomitantly, CD4 and CD8 cells of the UCP2-deficient mice showed increased production of reactive oxygen species. These results suggest a protective function of UCP2 in chronic inflammatory diseases such as multiple sclerosis.

Figure 1

Role of UCP2 in EAE. Quantification of UCP2 protein expression in spleen lymph nodes (LN) (A) and of UCP2 mRNA in LN and spinal cord (SC) (B) at different time points after the immunization of C57BL/6J. For UCP2 protein quantification, the relative UCP2 expression was normalized with the mitochondrial porin. Data are expressed as mean ± SEM of five mice per time point. UCP2 mRNA expression levels in LN and SP were measured by quantitative real-time polymerase chain reaction. The relative UCP2 expression was normalized with the GAPDH expression. Data are expressed as mean ± SEM of three mice per time point, measured in triplicate. C: Clinical scoring data from UCP2-deficient mice and the control group. C57BL/6J-UCP2^−/− (circle) and C57BL/6J (square) mice were scored for EAE lesions as described in Materials and Methods. The data presented are a combination of two independent experiments (n = 40 and 25 for UCP2^−/− and controls, respectively). *P < 0.05, **P < 0.001.

Figure 2

Morphological analysis of spinal cord sections in TMAs. A: Overview of the spinal core cylinder arrangement in the TMA, single spinal cord core (hematoxylin and eosin, 4× objective, 1.3 mm diameter). B: CD3 (T-lymphocyte infiltration, ×20) high-infiltration example. C: CD3 (T-lymphocyte infiltration, ×20) low-infiltration example. D: B220 (B-lymphocyte infiltration, ×20) (consecutive slide of B). E: IBA1 stain for microglia infiltration (×60).

Figure 3

Analysis of the immune response in UCP2^−/− and UCP2^+/+ mice. T-cell proliferation (A) and B-cell response (B) in UCP2 knockout mice (gray columns) and the control (white columns). Data are presented as mean ± SEM of n = 12 in the case of UCP2-deficient mice and n = 8 for the wild type. Statistical differences between the groups were analyzed using Mann-Whitney test. C: Cytokine productions in CD4 cells from immunized mice. The data presented are a combination of three independent experiments (n = 20 and 10 for UCP2^−/− and controls, respectively). *P < 0.05, **P < 0.001.

Figure 4

Comparison of the ROS production between the UCP2-deficient mice (gray columns) and the control (white columns) in T cells (A) and macrophages (B). Data are as mean ± SEM of 12 mice per group. *P < 0.05.