The antioxidant Tempol reduces CNS autoimmune disease in a model of multiple sclerosis

Multiple sclerosis is a chronic, inflammatory, demyelinating disease of the central nervous system. MS affects 300,000 young adults in the US with a 3:1 bias for women. Current therapies have moderate efficacy in targeting the immune response, but very few address the neurodegenerative component as well. Experimental allergic encephalomyelitis (EAE) is a mouse model for multiple sclerosis. In C57BL/6 mice, EAE is mediated by CD4+ T cells specific for the myelin protein MOG35-55 and causes an autoimmune response and demyelination. Symptoms are similar to chronic progressive MS and include weakness, spasticity, and eventually paralysis.

Oxidative stress is an established component of EAE and is suspected to be a major contributor to the disease process in MS. During an inflammatory response, macrophages and microglia produce nitric oxide (Gonsette, 2008). Nitric oxide combines with superoxide to create peroxynitrite (Beckman, etl al., 1990). Peroxynitrite can lead to radical damage of tissue and the inactivation of mitochondrial proteins (Strong, et. al., 2003). When the membrane potential and complexes of the mitochondria are altered, not enough ATP is produced to pump sodium out of the neuron. As the axon is demyelinated, more sodium channels are recruited to compensate, adding more intracellular sodium. To pump excess sodium out, the sodium/calcium exchanger works backward. The increase in intracellular calcium can lead to more peroxynitrite and to neuronal cell death (Andrews, et. al., 2005).

Tempol is a low molecular weight, hydrophilic, metal-free, non-toxic nitroxide (Mitchell, et. al, 1990). These qualities allow it to work intracellularly at high concentrations. Work in the lab has shown that Tempol can cross the blood brain barrier, Tempol fed mice with EAE have less neurofilament loss than control EAE mice, and mice have less severe physical disability when fed Tempol prophylactically or therapeutically (unpublished results). Tempol works as a peroxidase and superoxide dismutase mimic (Soule, et. al., 2007), inhibits lipid peroxidation (2007), and inhibits the Fenton reaction formation of hydroxyl radicals (Mitchell, et. al., 1990).

The purpose of this experiment was to determine whether Tempol influenced the development of pathogenic autoreactive T cells responsible for driving disease. Tempol's effects on the development, differentiation, and pathogenicity (ability to cause disease) of T cells were studied through analysis of T cell proliferation and cytokine production by thymidine incorporation, and enzyme-linked immunosorbent assay (ELISA) and intracellular cytokine fluorescent activated cell sorting (FACS), respectively.

The generation of antigen specific T cells was not reduced in the presence of Tempol. The supernatants of the proliferation were tested for IFN and IL-17. TH1 cells produce IFN . TH17 cells, which are currently thought to be the major mediator of EAE, produce IL-17 (Bettelli, et. al., 2008). Tempol fed mice produced slightly more IFN and IL-17 than control mice. Since Tempol did not reduce proliferation and actually increased IL-17 production, it protection is not through T cell mediation. Tempol more likely prevents myelin and axonal loss by inhibiting the formation and effects of free radicals. Its beneficial action may also depend on its interaction with iron, which is currently being studied in connection with MS. Tempol may be a powerful tool in EAE and MS because it may act to not only decrease inflammation but also prevent neurodegeneration.