Inhibition of TLR8- and TLR4-induced Type I IFN induction by alcohol is different from its effects on inflammatory cytokine production in monocytes

Abstract

Background: Prolonged alcohol consumption is a significant co-factor in the progression of chronic viral infections including hepatitis C and HIV, which are both single-stranded RNA viruses. Toll like receptor 8 (TLR8), a pattern recognition receptor expressed in monocytes, senses viral single stranded RNA as a danger signal and leads to the induction of Type I interferon (IFN) as well as the pro-inflammatory cytokine, tumor necrosis factor alpha (TNF alpha). Lipopolysaccharide (LPS), a Toll like receptor 4 (TLR4) ligand, was shown to affect inflammatory cell activation after alcohol consumption and in HIV and HCV infections. Here we hypothesized that alcohol exposure modulates TLR8- and TLR4-ligand-induced monocyte activation and affects both type I IFN and inflammatory cytokine induction.

Results: The TLR8 ligand, CL075, as well as the TLR4 ligand, LPS, resulted in a significant induction of TNF alpha both at the mRNA and protein levels in human monocytes. We found that both acute and prolonged alcohol treatment resulted in inhibition of type I IFN induction by either TLR8 or TLR4 ligands in human monocytes at the protein and mRNA levels. In contrast to Type I IFN production, the effects of acute and prolonged alcohol were different on inflammatory cytokine activation after TLR8 or TLR4 ligand stimulation. Acute alcohol inhibited TLR8- or TLR4-induced TNF alpha protein and mRNA induction while it augmented IL-10 production in monocytes. In contrast, prolonged alcohol treatment augmented TNF alpha without affecting IL-10 production significantly in response to either TLR8 or TLR4 ligand stimulation.

Conclusions: These novel results suggest first, that alcohol has a profound inhibitory effect on Type I IFN induction regardless of intracellular (TLR8) or cell surface-derived (TLR4) danger signals. Second, both acute and prolonged alcohol exposure can inhibit antiviral Type I IFN pathway activation. Third, the opposite effects of acute (inhibitory) and prolonged alcohol (augmentation) treatment on pro-inflammatory cytokine activation extend to TLR8-induced signals beyond the previously shown TLR4/LPS pathway.

Figure 1

Decreased IFNβ levels in monocytes stimulated with acute alcohol and TLR8 or TLR4 ligand. Human monocytes (n = 3) were isolated as described in the methods. Cells were either treated or not with 25 mM ethanol or 2.5 ug/ml CL075 (TLR8 ligand) or 0.1 ug/ml LPS (TLR4 ligand) for 6 hours. Some cells were stimulated with a combination of 25 mM ethanol and 2.5 ug/ml CL075 or 25 mM ethanol and 0.1 ug/ml LPS. The cell-free supernatants were collected and IFNβ production was measured by ELISA. Error bars represent ± SEM. unst: unstimulated, ns: non significant.

Figure 2

Prolonged alcohol exposure down regulates TLR8- or TLR4- induced IFNβ expression both at protein and mRNA levels. Human monocytes (n = 5 or 6) were either treated or not with 25 mM ethanol for 7 days, 2.5 ug/ml CL075 or 0.1 ug/ml LPS for 6 or 24 hours and alcohol treated cells were further stimulated or not with CL075 or LPS for 6 hours (A and C) or 24 hours (B). The cell-free supernatants were collected and analyzed for IFNβ production by ELISA (A and B, n = 6). Total RNA was extracted from the cells and real time PCR was performed to quantify the IFNβ gene expression (C, n = 5). Error bars represent ± SEM. unst: unstimulated, ns: non significant.

Figure 3

Acute alcohol treatment down regulates TLR8- or TLR4- induced TNF alpha production. Human monocytes (n = 3) were either treated or not with 25 mM ethanol or 2.5 ug/ml CL075 (TLR8 ligand) or 0.1 ug/ml LPS (TLR4 ligand) for 6 hours. Some cells were stimulated with a combination of 25 mM ethanol and 2.5 ug/ml CL075 or 25 mM ethanol and 0.1 ug/ml LPS. The cell-free supernatants were collected and TNF alpha production was measured by ELISA. Error bars represent ± SEM. unst: unstimulated.

Figure 4

Prolonged alcohol exposure augments TLR8 or TLR4 induced TNF alpha expression. Human monocytes (n = 6 or 7) were either treated or not with 25 mM ethanol for 7 days, 2.5 ug/ml CL075 or 0.1 ug/ml LPS for 6 or 24 hours and alcohol treated cells were further stimulated or not with CL075 or LPS for 6 hours (A and C) or 24 hours (B). The cell-free supernatants were collected and analyzed for TNF alpha production by ELISA (A and B, n = 6). RNA was extracted from the cells and real time PCR was performed to quantify TNF alpha gene expression (C, n = 5). Error bars represent ± SEM. unst: unstimulated.

Figure 5

Acute alcohol exposure augments TLR8- or TLR4- induced IL-10 expression. Human monocytes (n = 3) were either treated or not with 25 mM ethanol or 2.5 ug/ml CL075 (TLR8 ligand) or 0.1 ug/ml LPS (TLR4 ligand) for 6 hours. Some cells were stimulated with a combination of 25 mM ethanol and 2.5 ug/ml CL075 or 25 mM ethanol and 0.1 ug/ml LPS. The cell-free culture supernatants were collected and IL-10 production was measured by ELISA. Error bars represent ± SEM. unst: unstimulated.

Figure 6

No significant changes in IL-10 expression after prolonged alcohol treatment. Human monocytes (n = 6 or 7) were either treated or not with 25 mM ethanol for 7 days, 2.5 ug/ml CL075 or 0.1 ug/ml LPS for 6 or 24 hours and alcohol treated cells were further stimulated or not with CL075 or LPS for 6 hours (A and C) or 24 hours (B). The cell free supernatants were collected and analyzed for IL-10 alpha production by ELISA (A and B, n = 6 or 7). RNA was extracted from the cells and real time PCR was performed to quantify IL-10 gene expression (C, n = 5). Error bars represent ± SEM. unst: unstimulated. ns: non significant.