Phosphodiesterase 5 inhibition at disease onset prevents experimental autoimmune encephalomyelitis progression through immunoregulatory and neuroprotective actions

In addition to detrimental inflammation, widespread axon degeneration is an important feature of multiple sclerosis (MS) pathology and a major correlate for permanent clinical deficits. Thus, treatments that combine immunomodulatory and neuroprotective effects are beneficial for MS. Using myelin oligodendrocyte glycoprotein peptide 35-55 (MOG)-induced experimental autoimmune encephalomyelitis (EAE) as a model of MS, we recently showed that daily treatment with the phosphodiesterase 5 (PDE5) inhibitor sildenafil at peak disease rapidly ameliorates clinical symptoms and neuropathology (Pifarre et al., 2011). We have now investigated the immunomodulatory and neuroprotective actions of sildenafil treatment from the onset of EAE when the immune response prevails and show that early administration of the drug prevents disease progression. Ultrastructural analysis of spinal cord evidenced that sildenafil treatment preserves axons and myelin and increases the number of remyelinating axons. Immunostaining of oligodendrocytes at different stages of differentiation showed that sildenafil protects immature and mature myelinating oligodendrocytes. Brain-derived neurotrophic factor (BDNF), a recognized neuroprotectant in EAE, was up-regulated by sildenafil in immune and neural cells suggesting its implication in the beneficial effects of the drug. RNA microarray analysis of spinal cord revealed that sildenafil up-regulates YM-1, a marker of the alternative macrophage/microglial M2 phenotype that has neuroprotective and regenerative properties. Immunostaining confirmed up-regulation of YM-1 while the classical macrophage/microglial activation marker Iba-1 was down-regulated. Microarray analysis also showed a notable up-regulation of several members of the granzyme B cluster (GrBs). Immunostaining revealed expression of GrBs in Foxp3+-T regulatory cells (Tregs) suggesting a role for these proteases in sildenafil-induced suppression of T effector cells (Teffs). In vitro analysis of splenocytes from sildenafil-treated animals showed down-regulation of Th1/Th2/Th17 responses while Tregs were up-regulated. Additionally, sildenafil treatment prevented MOG-specific IgG2b accumulation in serum. Taken together these data demonstrates that daily sildenafil treatment from the initiation of EAE symptoms prevents further clinical deterioration by stimulating immunomodulatory and neuroprotective mechanisms. Importantly, we also show here that sildenafil enhances the ability of human Tregs from healthy donors to down-regulate the proliferation of Teffs in vitro, strongly supporting the potential of sildenafil for therapeutic intervention in MS.