Effects of Alcohol on Innate Immune Responses of BV‐2 Microglial Cell Line without Type 7 Adenylyl Cyclase

Abstract

Alcohol consumption affects immune cell functions and induces neuroinflammation. The mechanism of ethanol's action on the immune system however, are not fully understood. Cyclic AMP (cAMP) is known for its immunosuppressive effects and also is required for proper development of the immune system. Previous work in our laboratory has demonstrated that ethanol enhances the activity of adenylyl cyclase (AC) in an isoform‐specific manner. Type 7 AC (AC7) is most enhanced by ethanol, and is highly expressed in immune cells including macrophages. Therefore, we hypothesize that AC7 plays an important role in alcohol's effects on innate immune functions of microglia and on neuroinflammation through a cAMP pathway. To test this hypothesis, we generated AC7 knockout (KO) cell lines from the mouse BV‐2 microglia cell line by CRISPR‐Cas9 mediated genome editing. A cAMP accumulation assay was used to confirm AC7 KO. The effects of ethanol on innate immune responses of BV‐2 cells with or without AC7 were examined by measuring phagocytosis and oxidative bursts via flow cytometry.The cAMP accumulation assay on wild type (WT) BV‐2 cells showed enhancement of cAMP production in the presence of prostaglandin‐E1 (PGE1). cAMP production was further enhanced by addition of ethanol, as well as an AC7 specific activator, sphingosine‐1‐phosphate (S1P). cAMP production in AC7 KO cells was drastically decreased and did not respond to either ethanol or S1P. The results suggest that AC7 is the dominant form of AC in the microglia. Phagocytic activity measured using fluorescently labeled bacteria was significantly suppressed in the presence of isopropanol (Iso) in WT BV‐2 cells, while Iso did not significantly change the phagocytic activity of AC7 KO cells. Ethanol further reduced phagocytic activity of WT cells in the presence of Iso. Ethanol alone did not suppress phagocytosis in WT BV‐2 cells, but significantly inhibited phagocytosis in AC7 KO cells. This latter difference in response was unexpected and may indicate that AC7 can protect microglia from ethanol‐induced inhibition of phagocytosis. The production of reactive oxygen species was measured using dihydrorhodamine 123. We did not observe any notable differences in oxidative burst between WT and AC7 KO BV‐2 cells. The results indicated that AC7 elimination altered ethanol effects on some of the immune functions of BV‐2 cells, which is consistent with our hypothesis. We plan to further examine innate immune functions including bacterial killing, nitric oxide production, and proinflammatory cytokine expression in AC7 KO cells.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.