University of North Carolina at Chapel Hill Graduate School
Doi
Abstract
Drug-induced liver injury (DILI) is the primary cause of acute liver failure in the United States and is responsible for a substantial number of drug failures both pre- and post-market. Over 1,000 drugs have been associated with DILI and as a result, understanding the mechanisms of toxicity has proved extremely difficult. A common theme across mechanisms is the involvement of the immune system in the etiology, exacerbation, or resolution of DILI. It has become apparent that events at the hepatocyte level prior to cell death may influence immune responses and mediate the outcome of DILI. Hepatocyte-derived exosomes (HDE) may constitute one such immunomodulatory signal. HDE are constitutively released lipid-bound particles that have the unique ability to pass through fenestrations in the sinusoidal endothelium and enter the systemic circulation, potentially delivering stress signals to local and distal immune cells. The current research sought to establish HDE as mediators of early immune responses in the absence of overt DILI. We present a comprehensive analysis of the kinetics, content, and immunologic activity of HDE from primary hepatocytes stressed by sub-toxic drug exposures. To our knowledge, this is the first report of content profiling and functional analysis using primary human HDE in the context of DILI. Studies in rats, rat hepatocytes, and human hepatocytes exposed to the prototypical hepatotoxicant acetaminophen (APAP) verified changes in the RNA content of HDE prior to overt injury. Next, HDE from control- and APAP-treated primary human hepatocytes were collected for content profiling and functional analysis. The global profiles of mRNA and miRNA in HDE shifted as a result of drug exposure. Lastly, the immunologic activity of HDE was examined by exposing monocytes to HDE from control- and APAP-treated primary human hepatocytes. Differential gene expression in pathways related to immune cell function and cholesterol metabolism were observed. Functionally, APAP HDE exposure resulted in sensitization of monocytes to LPS stimulation. miRNA profiles in APAP HDE suggested that monocyte gene expression may have been mediated directly by exosomal miRNA. These results demonstrate that HDE influence immune cells before overt hepatotoxicity and highlight the mechanistic relevance of HDE in mediating DILI outcomes.Doctor of Philosoph
