RESUMEN
Although Fasudil has shown therapeutic potential in EAE mice, the mechanism of action are still not fully understood. Here, we examined the immunomodulatory effect of Fasudil on encephalitogenic mononuclear cells (MNCs), and tested the therapeutic potential of Fasudil-treated MNCs in active EAE. Fasudil inhibited expression of CCL20 on T cells and migration of T cells, decreased CD4(+) IFN-γ(+) and CD4(+) IL-17(+) T cells, but increased CD4(+) IL-10(+) and CD4(+) TGF-ß(+) T cells. Fasudil reduced expression of CD16/32 and IL-12, while elevating expression of CD206, CD23, and IL-10. Fasudil also decreased levels of iNOS/NO, enhanced levels of Arg-1, and inhibited the TLR-4/NF-κB signaling and TNF-α, shifting M1 macrophage to M2 phenotype. These modulatory effects of Fasudil on T cells and macrophages were not altered by adding autoantigen MOG35-55 to the culture, i.e., autoantigen-independent. Further, we observed that, in vitro, Fasudil inhibited the capacity of encephalitogenic MNCs to adoptively transfer EAE and reduced TLR-4/p-NF-κB/p65 and inflammatory cytokines in spinal cords. Importantly, Fasudil-treated encephalitogenic MNCs exhibited therapeutic potential when injected into actively induced EAE mice. Together, our results not only provide evidence that Fasudil mediates the polarization of macrophages and the regulation of T cells, but also reveal a novel strategy for cell therapy in MS.
Asunto(s)
1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Encefalomielitis Autoinmune Experimental/terapia , Inmunomodulación/efectos de los fármacos , Macrófagos/efectos de los fármacos , Linfocitos T/efectos de los fármacos , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Animales , Arginasa/genética , Arginasa/inmunología , Tratamiento Basado en Trasplante de Células y Tejidos , Quimiocina CCL20/genética , Quimiocina CCL20/inmunología , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Femenino , Regulación de la Expresión Génica , Interleucina-10/genética , Interleucina-10/inmunología , Interleucina-12/genética , Interleucina-12/inmunología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Glicoproteína Mielina-Oligodendrócito , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/inmunología , Fragmentos de Péptidos , Cultivo Primario de Células , Receptores de IgG/genética , Receptores de IgG/inmunología , Transducción de Señal , Linfocitos T/inmunología , Linfocitos T/trasplante , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunología , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/inmunología , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/inmunologíaRESUMEN
AIM: Multiple sclerosis (MS) is a relapsing-remitting inflammatory demyelinating disease that requires long-term treatment. Although Rho kinase inhibitor Fasudil shows good therapeutic effect in experimental autoimmune encephalomyelitis (EAE), an animal model of MS, certain side effects may limit its clinical use. This study aimed at observing the therapeutic potential of Fasudil-modified encephalitogenic mononuclear cells (MNCs) via nasal delivery in EAE and exploring possible mechanisms of action. METHODS: Experimental autoimmune encephalomyelitis was induced with myelin oligodendrocyte glycoprotein 35-55 in C57BL/6 mice, and encephalitogenic MNCs were treated with Fasudil in vitro. Mice received 3 × 106 cells/10 µL per nasal cavity on day 3 and 11 postimmunization, respectively. RESULTS: Fasudil-modified MNCs reduced clinical severity of EAE, improved demyelination, and decreased inflammatory cells in spinal cords. Immunohistochemical results indicated that CD4+ T cells and CD68+ macrophages were barely detected in Fasudil-MNCs group. Fasudil-modified MNCs decreased CD4+ IFN-γ+ and CD4+ IL-17+ T cells, increased CD4+ IL-10+ T cells, restrained M1 markers CD16/32, CCR7, IL-12, CD8a, enhanced M2 markers CD206, CD200, CD14 in spleen. Fasudil-modified MNCs inhibited the activation of inflammatory signaling p-NF-kB/P38, accompanied by the decrease of COX-2 and the increase of Arg-1 in spinal cord, as well as the reduction of IL-17, TNF-α, IL-6 and the elevation of IL-10 in cultured supernatant of splenocytes. Fasudil-modified MNCs enhanced the levels of neurotrophic factors BDNF and NT-3 in spinal cord. CONCLUSION: Our results indicate that intranasal delivery of Fasudil-modified MNCs have therapeutic potential in EAE, providing a safe and effective cell therapeutic strategy to MS and/or other related disorders.