In vivo studies of the mouse cerebral cortex have suggested that ethanol induces neuronal damage through neuroinflammation, specifically through the activation of the inflammasome–mediated pathway [5, 6]. To assess whether ethanol activates the same pathway in human cells in vitro, we evaluated the expression of NLRP3 and Casp1 in ethanol-treated and untreated iPS cells (Fig. 1c–e). We showed by IHC and Western Blot analysis that the amounts of activated Casp1 and NLRP3 were increased in iPS cells after ethanol exposure (Fig. 1d). Conversely, the levels of pro-caspase- 1 (p45) were not significantly altered (Fig. 1d), suggesting that ethanol drives the activation of Casp1 with no substantial effect on gene expression. We then evaluated the expression of cleaved Casp3, a marker for apoptosis and found a significantly increased number of Casp3-positive cells, as well as an enhanced Casp3 protein level in ethanol-exposed iPS cells (Fig. 1e and f). However, this ethanol-induced increase in apoptotic markers is not accompanied by any long-term changes in iPS cell proliferation rate (Fig. 1b). These data suggest that ethanol exposure in human iPS cells mediates an increase in inflammatory responses.
