To investigate the mechanisms of ethanol proinflammatory responses we measured the expression of TLR3 and HMGB1, a ubiquitous TLR3 co-agonist. Ethanol treatment increased brain TLR3 mRNA (Figure 8A-a). Toll-like receptor positive immunoreactivity (+IR) provides insight into protein levels and ethanol increased TLR3 + IR cells by at least 2 fold in cortex (Figure 8 A-b). Cells with upregulated TLR3 expression appear to be neurons, which are consistent with previous findings by others that TLR3 was expressed in many cell types of the brain in mice [18], including neurons [41], microglia [9] and astrocytes [23]. These measurements were assessed 24 hours after the last ethanol dose indicating that TLR increases persist after blood ethanol concentrations return to zero and during the poly I:C responses. HMGB1 can bind to and activate multiple TLR receptors [42] being required for TLR3 receptor activation [22]. Ethanol treatment increased both brain HMGB1 mRNA (Figure 8B-a) and HMGB1 + IR (Figure 8B-b) by about 2 fold in cortex. Thus, ethanol treatment increased both HMGB1 and TLR3 receptors in brain (Figure 8).
