Alcohol effects on immune responses of classical pro-inflammatory macrophages

Abstract

Abstract Alcohol consumption modulates immune responses. However, the mechanisms of ethanol’s action on the immune system are not fully understood. Previous studies demonstrated that ethanol enhances the activity of adenylyl cyclase (AC) in an isoform-specific manner, that type 7 AC (AC7) is most enhanced by ethanol, and that AC7 is highly expressed in immune cells. Therefore, we hypothesized that AC7 is a central player in regulating alcohol’s effects on immune responses. To examine this hypothesis, we generated AC7 knockout (KO) cell lines from a mouse microglia cell line, BV-2, and isolated primary bone marrow-derived macrophages (BMDM) from myeloid lineage-specific AC7 KO mice. Cells were activated by lipopolysaccharides (LPS) and interferon gamma (IFN-γ) to classical pro-inflammatory macrophages. The effects of ethanol on nitric oxide (NO) production of macrophages were examined via Griess reagent. LPS and IFN-γ synergistically increased nitric oxide (NO) production in wild type (WT) BV-2 cells in a dose-dependent manner. Ethanol suppressed LPS and IFN-γ co-activated NO production. AC7 KO BV-2 cells did not respond to LPS or IFN-γ activation. LPS increased NO production of both WT and AC7 KO BMDM. NO production of KO BMDM was significantly lower than that of WT BMDM. IFN-γ increased LPS-induced NO production of both WT and AC7 KO BMDM. Ethanol suppressed LPS-induced NO production in a dose-dependent manner in WT BMDM, while this effect was reduced in AC7 KO BMDM. The results suggest that AC7 is a key factor in the regulation of NO production and that the effect of ethanol on NO production depends on AC7. In the future, we will investigate the iNOS expression signaling cascade of classical pro-inflammatory macrophages in the presence of ethanol.