Human Binge Alcohol Intake Inhibits TLR4–MyD88 and TLR4–TRIF Responses but Not the TLR3–TRIF Pathway: HspA1A and PP1 Play Selective Regulatory Roles

Abstract

Binge/moderate alcohol suppresses TLR4–MyD88 proinflammatory cytokines; however, alcohol’s effects on TLR–TRIF signaling, especially after in vivo exposure in humans, are unclear. We performed a comparative analysis of the TLR4–MyD88, TLR4–TRIF, and TLR3–TRIF pathways in human monocytes following binge alcohol exposure. Mechanistic regulation of TLR–TRIF signaling by binge alcohol was evaluated by analyzing IRF3 and TBK1, upstream regulator protein phosphatase 1 (PP1), and immunoregulatory stress proteins HspA1A and XBP-1 in alcohol-treated human and mouse monocytes/macrophages. Two approaches for alcohol exposure were used: in vivo exposure of primary monocytes in binge alcohol–consuming human volunteers or in vitro exposure of human monocytes/murine macrophages to physiological alcohol concentrations (25–50 mM ethanol), followed by LPS (TLR4) or polyinosinic-polycytidylic acid (TLR3) stimulation ex vivo. In vivo and in vitro binge alcohol exposure significantly inhibited the TLR4–MyD88 cytokines TNF-α and IL-6, as well as the TLR4–TRIF cytokines/chemokines IFN-β, IP-10, and RANTES, in human monocytes, but not TLR3–TRIF–induced cytokines/chemokines, as detected by quantitative PCR and ELISA. Mechanistic analyses revealed TBK-1–independent inhibition of the TLR4–TRIF effector IRF3 in alcohol-treated macrophages. Although stress protein XBP-1, which is known to regulate IRF3-mediated IFN-β induction, was not affected by alcohol, HspA1A was induced by in vivo alcohol in human monocytes. Alcohol-induced HspA1A was required for inhibition of TLR4–MyD88 signaling but not TLR4–TRIF cytokines in macrophages. In contrast, inhibition of PP1 prevented alcohol-mediated TLR4–TRIF tolerance in macrophages. Collectively, our results demonstrate that in vivo and in vitro binge alcohol exposure in humans suppresses TLR4–MyD88 and TLR4–TRIF, but not TLR3–TRIF, responses. Whereas alcohol-mediated effects on the PP1–IRF3 axis inhibit the TLR4–TRIF pathway, HspA1A selectively suppresses the TLR4–MyD88 pathway in monocytes/macrophages.