O002 A novel function of interferon beta in promoting the generation of anti-inflammatory M2-like microglia in vitro and in vivo

Abstract

IntroductionIFNβ is an approved therapeutic option for the treatment of the autoimmune multiple sclerosis (MS). The molecular mechanisms underlying the effects of IFNβ in MS ant its animal model experimental autoimmune encephalomyelitis (EAE) are not fully understood and especially the effects of IFNβ in the CNS cells are largely unknown. In this study, we identified a novel mechanism for the beneficial effect of IFNβ in MS/EAE through the conversion of microglia (MG) from pro-inflammatory M1-like MG to anti-inflammatory M2-like MG. MethodsFor in vitro study: Neonatal MG were generated and activated with LPS (1μg/ml) in the presence or absence of IFNβ (1000U/ml) for 3h and 6h. Cells were then harvested and subjected to RNA extraction, followed by Q-PCR for IL-1β, IL-12p40, IL-23p19, TNFα and IL-10. To measure secreted cytokine production, the supernatants from cells treated with LPS in the presence or absence of IFNβ for 24h were collected and subjected to ELISA. To determine the effect of IFNβ-treated MG on Th1 and Th17 activation, MG matured with LPS in the presence or absence of IFNβ were co-cultured with T cells purified from spleens of 2D2 mice for 3days. The production of IFNγ and IL-17 was measured by both intracellular staining and ELISA.For in vivo study: Mice (n=12) were immunized with MOG35-55 and administrated with either IFNβ (10,000U/ml, n=6) or vehicle (n=6) every other day starting from day 5 after immunization. Clinical scores were recorded at day 15. Spinal cords and brains from EAE mice were subjected to Iba-1 immunostaining or RNA extraction, followed by Q-PCR for Arginase-1, YM-1 and IL-10 expression. ResultsOur results show that the expression and production of proinflammatory M1-type cytokines, such as IL-1β, IL-12p40, IL-23 and TNFα were inhibited by IFNβ and the production of anti-inflammatory M2 cytokine IL-10 was enhanced by IFNβ in LPS-stimulated primary MG. Through this phenotype switch, IFNβ-treated MG suppressed Th1 and Th17 activation leading to the downregulation of IFNγ and IL-17 production. In vivo, IFNβ-treated EAE mice exhibit lower clinical scores, reduced Iba-1 expression, and increased expression of the M2 markers, arginase, YM-1 and IL-10 in the CNS. ConclusionTaken altogether, our results demonstrate for the first time that IFNβ favors the generation of MG2 leading to the secretion of the anti-inflammatory cytokine IL-10 and reduced expression of the pro-inflammatory cytokines, IL-12, IL-23, IFNγ and IL-17 that provides a novel molecular mechanism for the beneficial effect of IFNβ in MS/EAE.