Retinoids: The Link Between Alcohol and Interferon?
Authors: Steven A. Weinman, Irina Tikhanovich
Journal: Hepatology (2016)

Potential conflict of interest: Nothing to report. See Article on Page 1783 One of the greatest accomplishments in the history of the field of hepatology is the 25‐year effort that led from the discovery of the hepatitis C virus (HCV) to the understanding of its life cycle and ultimately the development of highly effective targeted therapy. Perhaps less well appreciated is the extent to which the global scientific effort against HCV has transformed our knowledge of liver disease, host‐viral interactions, and antiviral immune responses. Among these insights are the understanding of the reversibility of cirrhosis, the prognostic interpretation of liver histology, and the genesis of hepatocellular carcinoma. However, of all the scientific fruits of HCV research, none have been as significant as the profound insights gained into the nature of the antiviral interferon (IFN) pathway and the factors that determine its efficacy in different individuals. Although the development of new anti‐HCV drugs has reached a state of maturity, basic scientific understandings of the nature of the liver‐virus interaction continue to emerge. One of the central issues that remain unresolved is the relationship between HCV and alcohol consumption. It is well documented that HCV and alcohol each exacerbate liver injury caused by the other, but even more profound is the observation that alcohol consumption leads to a more than seven‐fold increase in the rate of virological failure of IFN treatment.1 In spite of these strong clinical data, the mechanisms responsible for this effect are poorly understood—and their relevance, if any, to IFN‐free treatment regimens is unknown. Interferons generated by hepatocytes in response to viral infection both clear virus from infected cells and make uninfected cells resistant to infection. In the case of HCV, the viral RNA is recognized by the pattern receptor and RNA helicase, RIG‐I, and this triggers a signaling cascade involving the adapter protein MAVS, the downstream kinases TBK1 and IKKε, and the transcription factors IRF3 and IRF7. In hepatocytes, this results in the secretion of IFN‐β. This process engages the effector arm of the IFN pathway where type 1 IFN binding to the receptors INFAR1 and IFNAR2 results in activation of the receptor‐bound kinases TYK2 and JAK1. These in turn cause tyrosine phosphorylation of STAT1 and STAT2, which then form a heterotrimeric complex of pSTAT1, pSTAT2, and IRF9. This IFN‐stimulated gene factor 3 is the active transcription factor that binds many IFN‐sensitive response elements (ISREs) inducing IFN‐stimulated genes (ISGs) that suppress viral replication by multiple mechanisms. Prior to the development of direct‐activing antivirals, HCV therapy was almost wholly based on trying to activate this endogenous method to clear the virus. Endogenous IFN responses still determine the outcome of acute infections and may even play some role in direct‐activing antiviral efficacy. Initially, there was little understanding of how alcohol affected IFN responses, but as replicon and in vitro infectious virus models of HCV became available, multiple effects were observed. On one hand, lipid peroxidation, as caused by alcohol, is a potent inhibitor of HCV replication through direct effects on viral proteins.2 On the other hand, alcohol also suppresses the IFN pathway by reducing STAT1 tyrosine phosphorylation,3 suppressing permissive arginine methylation of STAT1,4 decreasing expression of STAT1 and STAT2, and inducing the accumulation of two suppressors of cytokine signaling, SOCS‐2 and SOCS‐3.5 Nonetheless, whether these mechanisms are sufficient to explain the effects of alcohol on viral clearance has never been established. In this issue of Hepatology, Cho et al.6 present some startling new findings that shed new light on the mechanism of alcohol‐induced suppression of the IFN response. They report that the Huh7 cell line generally used for HCV studies lacks expression of most alcohol metabolizing enzymes. They subsequently discovered that simply expressing alcohol dehydrogenase (ADH), by itself, suppressed genome replication of HCV. Among the known substrates of ADH is retinol, and they were able to show that the ability of different ADH isoforms to inhibit virus correlated with their ability to oxidize inactive retinol to the active nuclear receptor ligand, retinoic acid. This result was intriguing due to the long‐standing recognition that vitamin A and its derivatives are crucial in immune responses.7 Retinoid deficiency causes altered antibody responses, reduces B‐ and T‐cell proliferation, reduces cytolytic activity of natural killer cells, alters myeloid cell cytokine production, and impairs differentiation of immunoglobulin A+ secreting cells. In addition, there is substantial clinical literature relating vitamin A to HCV. Vitamin A levels are low in chronic hepatitis C patients, and pilot studies have suggested that retinoic acid supplementation improves viral clearance. Unfortunately, this result could not be duplicated in a controlled trial.8 Alcohol induces the depletion of retinoid stores, and in cirrhosis there is profound loss of hepatic vitamin A storage as a result of stellate cell activation. Retinoids also appear to play a role in clearance of other viruses through up‐regulation of RIG‐I,9 expression of type I IFN receptor, and up‐regulation of ISGs in uninfected bystander cells.10 Knowing this, Cho et al.6 examined whether ADH‐dependent changes in retinoids could explain their anti‐HCV effects. Through a series of gene expression studies, the authors were able to show that ADH expression activated multiple well‐defined retinoic acid responsive genes as well as retinoic acid response element (RARE) reporter activity. They then went on to analyze the impact of retinoic acid on IFN‐stimulated genes and made the novel observation that a large majority of known ISGs have RAREs within their promoter sequences. The addition of retinoic acid dramatically enhanced IFN‐induced ISG expression in primary hepatocytes and increased IFN's antiviral activity. This raised the intriguing possibility that ethanol's ability to inhibit IFN viral clearance resulted from the parallel ways in which retinol and ethanol are ultimately oxidized by ADH and then aldehyde dehydrogenase to the final products retinoic acid and acetic acid (Fig. 1). In this view, ethanol's suppression of the IFN response results from substrate competition between ethanol and retinol at the level of ADH. They obtained evidence to support this idea based on the differential effects of ADH isoforms that vary in their preference for ethanol over retinol.Figure 1: A schematic representation of the retinol‐ethanol substrate competition hypothesis. Retinol (ROL) and ethanol (EtOH) are competitive substrates for ADH, thus the presence of ethanol in the system reduces the steady‐state concentration of retinoic acid (RA). Retinoid‐specific promoter activity is required for optimal expression of IFN‐stimulated genes (ISGs) and antiviral effects. Abbreviations: ACH, acetaldehyde; ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenases; CRABP, cellular retinoic acid‐binding protein; HCV, hepatitis C virus; RAL, retinaldehyde; RAR/RXR, retinoic acid receptor/retinoid X receptor; RARE, retinoic acid response element. (Courtesy of Bo‐Ram Bang and Takeshi Saito.)This study comes with some caveats. The inability to directly measure retinoid levels leaves the possibility that substrate competition may not be the only mechanism for the ability of alcohol to suppress IFN signaling. In vitro studies measuring ISG expression and subgenomic replicon elimination may not reflect the situation in vivo, and the relative contributions of this RARE‐ISRE interaction as opposed to the earlier noted effects of ethanol on the JAK/STAT pathway have not been determined. Nonetheless, the demonstration of the strong regulatory function that retinoids have for ISG expression opens a new window into the control of IFN pathways and provides a new understanding of how IFN, retinoids, and alcohol interact. Conditions that deplete retinoids thus should impair a wide array of hepatic antiviral responses and raise questions that will require new investigation into the role of retinoids in other viral liver diseases and cirrhosis.