The Cytokine mRNA Increase Induced by Withdrawal from Chronic Ethanol in the Sterile Environment of Brain is Mediated by CRF and HMGB1 Release

BackgroundMany neurobiological factors may initiate and sustain alcoholism. Recently, dysregulation of the neuroimmune system by chronic ethanol (CE) has implicated Toll-like receptor 4 (TLR4) activation. Even though TLR4s are linked to CE initiation of brain cytokine mRNAs, the means by which CE influences neuroimmune signaling in brain in the absence of infection remains uncertain. Therefore, the hypothesis is tested that release of an endogenous TLR4 agonist, high-mobility group box 1 (HMGB1) and/or corticotropin-releasing factor (CRF) during CE withdrawal are responsible for CE protocols increasing cytokine mRNAs. MethodsAcute ethanol (EtOH; 2.75g/kg) and acute lipopolysaccharide (LPS; 250g/kg) dosing on cytokine mRNAs are first compared. Then, the effects of chronic LPS exposure (250g/kg for 10days) on cytokine mRNAs are compared with changes induced by CE protocols (15days of continuous 7% EtOH diet [CE protocol] or 3intermittent 5-day cycles of 7%EtOH diet [CIE protocol]). Additionally, TLR4, HMGB1, and downstream effector mRNAs are assessed after CE, CIE, and chronic LPS. To test whether HMGB1 and/or CRF support the CE withdrawal increase in cytokine mRNAs, the HMGB1antagonists, glycyrrhizin and ethyl pyruvate, and a CRF1 receptor antagonist (CRF1RA) are administered during 24hours of CE withdrawal. ResultsWhile cytokine mRNAs were not increased following acute EtOH, acute LPS increased all cytokine mRNAs 4hours after injection. CE produced no change in cytokine mRNAs prior to CE removal; however, the CE and CIE protocols increased cytokine mRNAs by 24hours after withdrawal. In contrast, chronic LPS produced no cytokine mRNA changes 24hours after LPS dosing. TLR4 mRNA was elevated 24hours following both CE protocols and chronic LPS exposure. While chronic LPS had no effect on HMGB1mRNA, withdrawal from CE protocols significantly elevated HMGB1mRNA. Systemic administration of HMGB1antagonists or a CRF1RA significantly reduced the cytokine mRNA increase following CE withdrawal. The CRF1RA and the HMGB1 antagonist, ethyl pyruvate, also reduced the HMGB1mRNA increase that followed CE withdrawal. ConclusionsBy blocking HMGB1 or CRF action during CE withdrawal, evidence is provided that HMGB1and CRF release are critical for the CE withdrawal induction of selected brain cytokine mRNAs. Consequently, these results clarify a means by which withdrawal from CE exposure activates neuroimmune function in the sterile milieu of brain.