title: Phosphodiesterase 5 inhibition at disease onset prevents experimental autoimmune encephalomyelitis progression through immunoregulatory and neuroprotective actions.
abstract: 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.
authors.affiliation: Institute of Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona Spain. Electronic address: paula.pifarre@gmail.com.
authors.forename: Paula
authors.initials: P
authors.lastname: Pifarré
authors.affiliation: Institute of Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona Spain. Electronic address: maria.gutierrez.mecinas@uab.cat.
authors.forename: María
authors.initials: M
authors.lastname: Gutierrez-Mecinas
authors.affiliation: Institute of Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona Spain. Electronic address: Judith.prado@uab.cat.
authors.forename: Judith
authors.initials: J
authors.lastname: Prado
authors.affiliation: Institute of Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona Spain. Electronic address: lorena.usero@uab.cat.
authors.forename: Lorena
authors.initials: L
authors.lastname: Usero
authors.affiliation: Institute of Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona Spain; Department of Cellular Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain. Electronic address: carme.roura@uab.cat.
authors.forename: Carme
authors.initials: C
authors.lastname: Roura-Mir
authors.affiliation: Institute of Neuroscience, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona Spain; Department of Cellular Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain. Electronic address: merce.giralt@uab.cat.
authors.forename: Mercedes
authors.initials: M
authors.lastname: Giralt
authors.affiliation: Institute of Neuroscience, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona Spain; Department of Cellular Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain. Electronic address: juan.hidalgo@uab.cat.
authors.forename: Juan
authors.initials: J
authors.lastname: Hidalgo
authors.affiliation: Institute of Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona Spain; Department of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona Spain. Electronic address: agustina.garcia@uab.cat.
authors.forename: Agustina
authors.initials: A
authors.lastname: García
mesh_terms: D000818:Animals; D001369:Axons / Q000187:drug effects / Q000473:pathology / Q000648:ultrastructure; D001921:Brain / Q000473:pathology; D016207:Cytokines / Q000378:metabolism*; D004195:Disease Models, Animal; D018450:Disease Progression; D004681:Encephalomyelitis, Autoimmune, Experimental / Q000139:chemically induced / Q000473:pathology / Q000517:prevention & control*; D005260:Female; D005620:Freund's Adjuvant / Q000633:toxicity; D005786:Gene Expression Regulation / Q000187:drug effects / Q000276:immunology*; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D004676:Myelin Basic Protein / Q000378:metabolism; D063308:Myelin-Oligodendrocyte Glycoprotein / Q000276:immunology / Q000633:toxicity; D009836:Oligodendroglia / Q000187:drug effects; D058986:Phosphodiesterase 5 Inhibitors / Q000627:therapeutic use*; D010879:Piperazines / Q000627:therapeutic use*; D011687:Purines / Q000627:therapeutic use; D012720:Severity of Illness Index; D000068677:Sildenafil Citrate; D013450:Sulfones / Q000627:therapeutic use*; D013601:T-Lymphocytes / Q000187:drug effects* / Q000378:metabolism; D013997:Time Factors
chemical_list: D016207:Cytokines; D004676:Myelin Basic Protein; D063308:Myelin-Oligodendrocyte Glycoprotein; D058986:Phosphodiesterase 5 Inhibitors; D010879:Piperazines; D011687:Purines; D013450:Sulfones; D005620:Freund's Adjuvant; D000068677:Sildenafil Citrate
keywords: EAE; Immunomodulation; Neuroprotection; Phosphodiesterase 5; T regulatory cells