Alcohol use disorder (AUD) contributes significantly to disease burden and mortality in the United States, with an annual healthcare cost of $223 billion and a death toll of nearly 88,000. Current methods to assess alcohol intake rely primarily on self-report surveys or biochemical measures of liver injury. However, these methods are rather insensitive and unreliable since subjects are often not forthcoming and liver injury is not the only indication of ethanol-mediated organ damage, which includes increased incidence of gastrointestinal cancers and exacerbation of diabetes and cardiovascular disease. Moreover, alcohol consumption affects human health in a dose-dependent manner; light to moderate drinkers have a lower risk of all-cause mortality than abstainers while heavy drinkers are at the highest risk. Similarly, alcohol consumption modulates immunity in a dose-dependent manner. Specifically, AUD results in poor response to vaccination, enhanced susceptibility to infection, and poor wound healing, whereas moderate ethanol consumption leads to improved vaccine responses. Therefore, there is an urgent need to develop sensitive, robust, non-invasive markers of ethanol consumption. Extracellular RNA (exRNA) serves as efficient biomarkers of cancer, cardiovascular risk, liver injury and placental dysfunction. These exRNA circulate either packaged within exosomes, or bound to lipoproteins or other proteins, making them very stable. Given the role cellular miRNAs play in regulating gene expression following alcohol exposure, their potential to provide reliable biomarkers of ethanol consumption is high. However, our current understanding of alcohol-induced changes in exRNA is limited to a few exosome- bound microRNAs, which are increased in circulation in alcoholic liver disease models or after in vitro treatment of monocytes with rather high doses of ethanol. Thus, there is a crucial need to uncover additional dose-sensitive exosome-bound miRNA as well as identify miRNA cargo bound to protein and lipoproteins. In this application, we will address this critical gap in knowledge by first identifying ethanol-mediated changes in extracellular microRNA (ex-miRNA) bound to exosomes, lipoproteins, and proteins by leveraging a robust nonhuman primate model of voluntary chronic (12 months) ethanol self-administration in combination with a novel RNA-Differential Isolation Platform (RNA-DIP) and next generation sequencing. Additionally, we will test the functional impact of over-represented miRNA packaged in exosomes and delivered into monocytes from ethanol nave animals. We will assess the downstream effect of miRNA uptake by stimulating the cells with lipopolysaccharide (LPS) and measuring both changes in transcription and protein levels of inflammatory mediators. These experiments will be carried out in collaboration with Dr. Grant who developed the voluntary ethanol self-administration model, Dr. Zhong who developed the RNA-DIP chip to simultaneously isolate all three exRNA fractions, and Dr. Zhao who bears expertise in manipulating stem cell behavior by delivering exosome-bound miRNA. Completion of the studies proposed in this application will yield unprecedented insight into ethanol dose-dependent alterations in exRNA cargo, their potential role as biomarkers of ethanol consumption, and their impact on inflammation.
Alcohol use disorder results in significant disease burden and mortality, but the mechanisms by which it leads to negative health outcomes are not well understood. In this application, we will carry out a comprehensive and unbiased characterization of circulating small RNA to identify sensitive biomarkers of ethanol consumption and evaluate their role in inflammation. These results will positively impact future studies by identifying pathways that can be targeted therapeutically to overcome the adverse effects of alcohol use disorder.
