To confirm the regulatory role of IRAK-M in acute and chronic alcohol-exposed monocytic cells, we either inhibited the IRAK-M using siRNA after acute alcohol exposure or overexpressed IRAK-M in chronic alcohol exposed macrophages. Fig. 3A shows that in acute alcohol-exposed macrophages with 70% knockdown of IRAK-M mRNA (upper panel) and protein (middle panel), significant restoration of LPS-induced TNF-α levels occurred. These data indicate that IRAK-M contributes to the reduction in LPS-induced TNF-α production during acute alcohol exposure. In contrast, efficient overexpression of IRAK-M indicated by western blot analysis (upper panel) in chronic alcohol exposed macrophages prevented up-regulation of LPS-induced TNF-α levels (Fig. 3B; lower panel) suggesting an important role for IRAK-M in increased proinflammatory responses during chronic alcohol exposure.
