Phytosterols as immune modulators of multiple sclerosis.

摘要

Phytosterols (PS) are plant-based dietary components which have recently been shown to elicit anti-inflammatory properties in cells derived from Multiple Sclerosis (MS) patients. MS is a debilitating autoimmune disorder characterized by inflammatory driven demyelination of the CNS tissues and resultant ascending paralysis. Experimental Autoimmune Encephalomyelitis (EAE) is the closest animal model which mimics the inflammatory driven pathogenesis similar to that of MS. Based on similarities between MS and the EAE model, the overall purpose of this thesis was to investigate the role of dietary PS as immune modulators of MS. Additionally we aimed to investigate some of the molecular mechanisms by which PS may be functioning as anti-inflammatory molecules. In the first study, mice were pretreated with PS via gavage one week prior to and throughout disease induction. Following disease induction, mice were scored for EAE clinical symptoms. Upon sacrifice, brains and splenocytes were used to measure infiltration of immune cells and inflammatory activity. In subsequent studies, murine macrophages were utilized in culture to determine the effect of SIT, the predominant PS in the diet, on the innate inflammatory response. Using LPS to elicit inflammation, our studies utilized western blot, sandwich ELISA, cell-based ELISA, ImageStream cytometry as well as many other immunological/biochemical assays to determine how SIT may function as an anti-inflammatory molecule. Results from the in vivo study revealed that pre-treatment with PS provided protection against the development and severity of EAE compared to controls. Moreover, this study found that with reduced incidence of disease, there was reduced infiltration of inflammatory cells in the brains of treated mice and subsequently, less demyelination. Also, inflammatory activity was markedly reduced in tissues derived from treated mice. Using macrophages in culture, the results of the second experiment found that SIT elicits anti-inflammatory activity by reducing activation of transcription factors NF-kappaB and STAT1. Along with these findings, our results indicated that there was a decrease in pro-inflammatory cytokines and chemokines and a marked increase in regulatory IL-10 and anti-inflammatory phosphatase SHP-1. Finally, our last in vitro study determined that NF-kappaB activity may be directly reduced in response to alterations in TLR4 pathway activity. Results from this study showed that SIT treatments altered expression of TLR4 on the surface of the cell membrane, but did not alter the expression of lipid rafts or co-localization of the receptor into the rafts. Moreover, looking at intracellular pathways, SIT significantly decreased MyD88 and pIRAK1 expression compared to dose-matched cholesterol treatments and control. Finally, SIT treatment up-regulated the expression of the regulatory protein SOCS3 in response to LPS stimulation. Taken together, our results from both in vivo and in vitro studies provide evidence that PS elicit anti-inflammatory properties. Since PS are dietary components, these data suggest that increasing their consumption may be beneficial as a protective agent against development of inflammatory based disease.