We previously demonstrated that changing the timing and extent of activation or the differentiation path of microglia changed the outcomes of EAE. Here, we examined nicotine’s effect on microglia in the context of these states and found that nicotine significantly delayed and inhibited microglial activation during EAE, and that this inhibition primarily proceeded through suppression of M1 microglial differentiation. In culture, nicotine decreased TNF-a release from NCM-stimulated microglia. Previous studies demonstrated that M1 microglia promote T cell differentiation toward Th1 and Th17 fates, induce neurodegeneration and impair remyelination. These effects of M1 microglia were mediated primarily by TNF-a. Therefore, biased suppression of M1 microglia through nicotine administration supports an antiinflammatory environment, possibly re-balancing T cell populations, protecting neurons and myelin sheaths from insults, and promoting recovery of the tissues. It was reported that nicotine inhibited microglia/macrophage activation primarily through binding to a7nAChRs. Nevertheless, attenuation of EAE outcomes by nicotine was only partially reversed in a7 mice, which suggested that other nAChRs subunits might also contribute to the impact of nicotine on EAE. Ohnishi et al. showed that, besides a7, b2containing nAChRs were involved in nicotine. In addition, a5 subunits contributed to immune regulatory functions, because the absence of a5 increased severity of experimental colitis. Taken together, nicotine could potentially regulate microglia functions and EAE outcomes through targeting multiple nAChRs pathways. In contrast, we demonstrated that CSC had a detrimental effect on EAE BU 4061T progression. However, the significant effect was only achieved during early stages of EAE. This might be due to the fact that the inflammation in mice due to ongoing EAE is more severe than the inflammatory effects mediated by this concentration of CSC. The highest concentration of CSC that we could use in vivo was 20 mg/ml, which contains only 2–3% of nicotine. If we had been able to deliver significantly higher concentrations of CSC, then more severe EAE symptoms might have been encountered. Nonetheless, we used CSC-treated EAE samples and cell culture to explore how CSC affects inflammation. Our results indicate that CSC both activates microglia, and eventually becomes cytotoxic to the cells. Other studies have shown that low concentrations of CSC induce inflammatory cytokine release from pulmonary macrophages, while higher concentrations cause cell death. These reports support our findings that CSC enhances inflammation in EAE mice. We and others have previously shown that inhibition of microglia/macrophage function protects from EAE, further suggesting that CSC adversely affects EAE scores. Further, we have shown that inhibition of acrolein in CSC by hydralazine significantly reduces cell death in treated primary microglia.