Circulating microRNAs in exosomes indicate hepatocyte injury and inflammation in alcoholic, drug-induced, and inflammatory liver diseases

Abstract

MicroRNAs are fine tuners of diverse biological responses and are expressed in various cell types of the liver. Here we hypothesized that circulating microRNAs (miRNAs) may serve as biomarkers of liver damage and inflammation. We studied miRNA-122 that is abundant in hepatocytes and miR-155, -146a and -125b that regulate inflammation in immune cells in mouse models of alcoholic liver disease (ALD), drug (acetaminophen; APAP)-induced liver injury (DILI), and Toll-like receptor (TLR) 9+4 ligands-induced inflammatory cell-mediated liver damage. We found that serum/plasma miR-122 correlated with ALT increases in the liver damage caused by alcohol, APAP and TLR9 (CpG)+4 (LPS) ligands. MiR-155, a regulator of inflammation, was increased in serum/plasma in alcoholic and inflammatory liver injury. Alcohol failed to increase serum miR-122 in TLR4-deficient and p47phox-deficient mice that were protected from ALD. We found the most robust increase in plasma miR-122 in DILI and it correlated with the highest ALT levels. Consistent with the massive inflammatory cell infiltration in the liver, plasma miR-155 and miR-146a were significantly elevated after CpG+LPS administration. We show for the first time that, depending on the type of liver injury, circulating miRNAs show association either with the exosome-rich or protein-rich compartments. In ALD and in inflammatory liver injury, serum/plasma miR-122 and miR-155 were predominantly associated with exosome-rich fraction while in DILI/APAP injury these miRNAs were present in the protein-rich fraction. In conclusion, our results suggest that circulating miRNAs may serve as biomarkers to differentiate between hepatocyte injury and inflammation and the exosome versus protein association of miRNAs may provide further specificity to mechanisms of liver pathology.

MicroRNAs in Liver Disease: Bench to Bedside

“…115 A profound increase was found in serum miR-122 levels in alcohol-fed mice with a linear correlation between serum ALT and serum miR-122 levels. 21 MiR-27b, miR-214, miR199a-3p, miR-182, miR-183, miR-200a, and miR-322 were found to be down-regulated, whereas miR-705 and miR-1224 were increased after 4 weeks of alcohol feeding in mice. 116 Alcohol has been shown to down-regulate miR-199 in rat liver sinusoidal endothelial cells and human endothelial cells.…”

“…18 Acetaminophen administration results in increased miR-122, miR-155, miR-125b and miR-146a levels and these signatures could potentially represent biomarkers of acetaminopheninduced liver injury. 21 MiR-122 and -192 are shown to exhibit dose and exposure dependent changes in plasma parallel to aminotransferases and hepatocellular damage. 18 They were not only found to be significantly higher but also detected earlier in patients with acetaminopheninduced liver injury as compared with healthy controls.…”

“…20 Bala et al have shown that miR-122 and miR-155 were predominantly associated in protein aggregates in acetaminophen-induced liver necrosis, whereas in alcoholic liver disease associated inflammatory damage, these two miRs were mainly found in the exosome-rich vesicular fraction, suggesting an injuryspecific distribution of miRs in the circulation. 21 Circulating miRs have tremendous stability in extreme conditions including a low pH environment, they are also resistant to the high endogenous RNase activity present in serum due to protein aggregation and vesicle enclosure and are stable in serum incubated at room temperature for at least 24 h or subjected to up to at least eight freeze-thaw cycles. 17,22,23 For these reasons circulating miRs are promising, non-invasive diagnostic biomarkers for liver disease.…”

The Reliability of Plasma Exosome Concentrations in Healthy Male Individuals

“…Exclusion criteria included diagnosis of a chronic inflammatory condition (e.g diabetes mellitus and cardiovascular diseases or recent consumption of anti-inflammatory medications (e.g ibuprofen). Since the scope of the study is to study the variation found in the levels of exosomes or their concentration in healthy individuals, therefore the indication of any inflammatory condition refers to the natural increase in the concentration of exosomes and any proteins that are associated with anti-inflammatory response [20]. Moreover, subjects were excluded if they had any pathology known to influence immune response (e.g.…”

Simultaneous Enrichment of Plasma Soluble and Extracellular Vesicular Glycoproteins Using Prolonged Ultracentrifugation-Electrostatic Repulsion-hydrophilic Interaction Chromatography (PUC-ERLIC) Approach*

“…Although extracellular vesicles can be isolated using methods such as filtration concentration, flotation density gradients and immunocapture beads, the most widely used method is successive centrifugation followed by ultracentrifugation (UC) (23,24). Extracellular vesicles are known to be involved in myriad cellular processes including coagulation (25), inflammation (26), tumor progression (27), immune response regulation (28), ectodomain shedding of membrane proteins (29), antigen presentation (30), intracellular communication (31), transfer of RNA and proteins (32) and transfer of infectious cargo (prions and retroviruses) (33,34). In addition, extracellular vesicles have been implicated in the pathogenesis of vascular disorders (35,36), cancers (37,38), diabetes (39) and infectious diseases (40,41).…”