The immunomodulatory effects of alcohol use have been described in various clinical settings including bacterial and viral infections as well as in post-trauma and post-surgical recoveries [2–4, 34]. Increased activation of the inflammatory cascade was found in chronic alcoholics with liver disease where blood monocytes produced increased levels of the pro-inflammatory cytokine TNF alpha [35, 36]. While inflammatory cytokine activation has been investigated in several conditions related to alcohol consumption, little is known about the effects of alcohol on the induction of anti-viral immune mediators.
In this study, we investigated the effects of acute and prolonged alcohol on human monocyte responses to TLR8 and TLR4 ligand stimulation and evaluated anti-viral (Type I IFNs), pro- (TNF alpha) and anti-inflammatory (IL-10) cytokine production. Our novel data indicate that IFNβ production in response to TLR8 or TLR4 stimulation was significantly attenuated in monocytes exposed to alcohol. These data suggest that alcohol, regardless of the length of exposure, has inhibitory effects on Type I IFN production in monocytes. Our data further demonstrate that the effects of prolonged alcohol are opposite on Type I IFN induction and inflammatory cytokine induction within the same cell type. Prolonged alcohol exposure inhibited IFNβ production while it increased TLR8- and TLR4-induced TNF alpha production. Finally, we show that the previously described anti-inflammatory effect of acute alcohol on TNF alpha production extends to TLR8-induced TNF alpha induction and involves simultaneous induction of the anti-inflammatory cytokine, IL-10, in human monocytes.
Toll-like receptors are evolutionarily preserved receptors for recognition of danger signals . Nucleic acid sequences in viruses are recognized by TLRs expressed in the endosome including TLR7 and TLR8 that sense single-stranded RNA and TLR9 that sense bacterial DNA . LPS, a component of gram-negative bacteria, activates TLR4 expressed on the cell surface . Human monocytes express a broad repertoire of functionally active TLRs including TLR8 and TLR4 [9, 27]. We found that TLR8 or TLR4 ligand stimulation resulted in the induction of both IFNβ and inflammatory cytokines in human blood monocytes. Ligand activation of TLR8 recruits MyD88, a common TLR adapter, and triggers activation of IRAK1/4 kinases and the TRAF6/TAK1 complex leading to the activation of the IKK kinase complex that in turn induces inflammatory cytokines, TNF alpha or IL-10 or phosphorylation of IRFs that induce production of Type I IFNs . TLR4 signaling involves recruitment of the adaptor molecules MyD88 and/or TRIF, each activating different downstream pathways to induce inflammatory cytokines via NF-κB or Type I IFNs via IRF activation, respectively . Considering the differences between the TLR8- and TLR4-induced signaling, our data suggest that alcohol likely modulates multiple components of these signal transduction pathways. Indeed, modulation of NF-κB by acute and chronic alcohol has been extensively studied in the past in LPS-stimulated monocytes [28, 39].
The inhibitory effect of alcohol on Type I IFN induction is likely to be a clinically significant finding considering the large number of studies that demonstrate a clinical correlation between alcohol use and impaired anti-viral immune mechanisms [3, 17, 18, 40, 41]. Excessive alcohol consumption is known to predispose individuals to secondary infections such as HCV, HIV and bacterial infections [5–7, 20]. We found that the inhibitory effects of alcohol occurred both at the IFNβ protein and mRNA levels. As we found no significant changes in TLR8 and TLR4 mRNA or protein after alcohol treatment, we speculate that alcohol interferes with some of the intracellular signaling elements required for IFNβ gene induction in monocytes. Recently, Zhao et al have described the role of PPAR-gamma in limiting IFNβ production via targeting IRF3 in macrophages . In line with this report, alcohol has been shown to activate PPAR-gamma in rats  and mice .
Previous studies from our and other laboratories showed that IFN-induced signaling pathways are also inhibited by alcohol in blood monocytes as well as in liver cells [19, 39, 45–47]. Phosphorylation of STAT-1 and STAT-3 was inhibited by acute alcohol suggesting not only IFNβ production but even Type I IFN-induced downstream events could be impaired by alcohol exposure . Recently, we showed that chronic alcohol upregulates microRNA (miRNA)-155 in macrophages  therefore, it is reasonable to argue that alcohol might be targeting other miRNAs to limit IFNβ production. In fact, miR-26a, -34a, -145, and let-7b are shown to directly regulate IFNβ in human and macaque cells .
We found that acute and prolonged alcohol had opposite effects on both TLR8- and TLR4-induced TNF alpha production in human monocytes. Our laboratory has extensively studied the opposite effects of acute and prolonged alcohol on TLR4-mediated signaling pathways and identified IRAK-M, IRAK1, IKK phosphorylation and NF-κB activation as common targets of acute and chronic alcohol with opposite effects.
We showed that the inhibitory effects of acute alcohol are mediated by alcohol- induced inhibition of NF-κB activation due to decreased phosphorylation of IKKα and IKKβ in LPS-stimulated human monocytes . Based on this information and the fact that TNF alpha induction involves NF-κB activation whether induced via TLR8 or TLR4, our observation of decreased TNF alpha in acute alcohol-exposed TLR8 stimulated monocytes suggests that NF-κB inhibition is a likely mechanism for inhibition of TLR8- induced TNF alpha induction by acute alcohol. An additional mechanism that likely contributes to decreased TNF alpha production after acute alcohol is the increased induction of IL-10 observed in TLR8 or TLR4 stimulated alcohol-exposed monocytes in our experiments. The increased TNF alpha induction by TLR8 ligand in chronic alcohol-exposed monocytes might involve NF-κB activation and reduced IRAK-M by chronic alcohol is also permissive to increase TNF alpha production . Finally, induction of miR-155 after chronic alcohol treatment results in increased TNF alpha production via increasing TNF alpha mRNA stability , which is likely the mechanism not only for TLR4- but also TLR8-induced TNF alpha production in monocytes.
Lastly, alcohol treatment (acute or chronic) modulates cytokines that negatively regulate TNF alpha. Previous studies from our laboratory demonstrated that acute alcohol increases IL-10, TGFβ and IL-13 production in human monocytes [51–53]. In a mouse model, acute alcohol has been shown to inhibit TLR- induced inflammatory responses via the p38 and ERK1/2 pathway . While our or others studies found no obvious decrease in the level of anti-inflammatory cytokines after prolonged alcohol exposure in monocytes or in vivo, there is an obvious imbalance between pro- and anti-inflammatory cytokines with the presence of increased TNF alpha and no increase in IL-10 in human monocytes after prolonged alcohol treatment. Thus, it is possible that prolonged alcohol exposure targets multiple signaling molecules to augment TNF alpha expression.