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, which is abundant in hepatocytes, and miR-155, -146a, and -125b, which 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 ligand-induced inflammatory cell-mediated liver damage. We found that serum/plasma miR-122 correlated with alanine aminotransferase (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 are associated 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 the exosome-rich fraction, whereas in DILI/APAP injury these miRNAs were present in the protein-rich fraction.

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.

Fig. 1. Increased circulating miR-122 and miR-155 in alcoholic liver disease

Eight-week old C57BL/6 female mice received Liber De-Carli diet either with 5% alcohol (alcohol-fed) or isocaloric diet (pair-fed) for 5weeks. A. Hematoxylin and eosin (H&E) staining of liver sections fixed in formalin. Solid arrow indicates immune cells infiltrate, broken arrow indicates fat accumulation in hepatocytes and double arrow indicates mild necrosis of hepatocytes. B. ALT and miR-122 levels (TaqMan qRT-real time PCR) were measured in the serum as described in methods (n=6–8). C. Correlation between serum ALT and serum miR-122 was determined by Pearson method (n=30). D. The protein level of TNFα was measured from the whole cell lysate of liver homogenates by ELISA and normalized to protein concentration (n=6–8). E. MiR-155 expression was detected in the serum by TaqMan qRT-real time PCR (n=5). Synthetic C. elegans miR-39 was used to normalize Ct values. Fold change was calculated compared to pair-fed mice. Data represent mean ± SEM. Statistical analysis was performed with Two-tailed t-test (B&D) or non-parametric Mann-Whitney test (E).

Fig. 2. TLR4 deficiency or disruption of oxidative stress prevents increased serum miR-122 in ALD

Wild type (WT) or TLR4- or p47^phox - deficient mice (KO) were fed with isocaloric (pair-fed) or alcohol (ethanol-fed) containing Liber De-Carli diet for 5 weeks. A. H&E staining of formalin fixed liver sections from WT and TLR4KO mice. B. Serum ALT analysis of WT and TLR4KO mice (n=6–7). C. The expression of miR-122 in the serum isolated from WT and TLR4KO mice was quantified by TaqMan qRT-real time PCR (n=5–7). D. Histopathology (H&E) of liver sections from WT and p47^phox KO mice. E. Serum ALT analysis of WT and p47^phox KO mice (n=6–7). F. The expression of miR-122 in the serum was determined by TaqMan qRT-real time PCR in WT and p47^phox KO mice (n=6). Spiked C. elegans miR-39 was used to normalize Ct values. Fold change was calculated compared to pair-fed mice. Data represent mean ± SEM. Two-tailed T-Test (B,C&E) or non-parametric Mann-Whitney test (F) was employed for statistical analysis.

Fig. 3. APAP administration induces a time-dependent increase of miRNA in the plasma

Wild type female mice (8 weeks old) were fasted overnight as describes in methods and next day, some mice received saline or APAP (500mg/kg, lethal dose) for 3h or 6h. A. H&E staining of formalin fixed liver sections. Broken arrows indicate centrilobular cytoplasmic vacuoles, solid arrows indicate large cytoplasmic vacuoles and double arrows indicate extensive parenchymal hemorrhages. B. Plasma ALT levels (n=8). C. The expression of miR-122 in the plasma was quantified by TaqMan qRT-real time (n=8). D. Correlation between plasma ALT and miR-122 was determined by Pearson method (n=23). E-F. The relative expression of miR-155 (E), -146a (F left) and miR-125b (F right) in plasma was determined by TaqMan qRT-real time PCR (n=8). Spiked C. elegans miR-39 was used as internal control. Fold change over saline treated mice is shown. Data represent mean ± SEM. Non-parametric Mann-Whitney test was employed for statistical analysis.

Fig. 4. Increased plasma miRNA-122, -155 and -146a in CpG+LPS-induced liver inflammation

Wild type female mice (10–12 weeks old) were injected with 2.5mg/kg CpG DNA (i.p.) once a day for three days and on day 4 some mice received 0.5mg/kg LPS or saline for 3h and sacrificed. A. H&E staining of formalin fixed liver sections. Broken arrow indicates mononuclear inflammatory cells and solid arrows indicate inflammatory foci B. Plasma ALT levels (n=4–6). C. The expression of miR-122 in the plasma was quantified by TaqMan qRT-real time (n=4–6). D. Correlation between plasma ALT and miR-122 was determined by Pearson method (n=17). E. The expression of TNFα was measured by real time PCR using TNFα gene-specific primer and normalized to 18S. F. The relative expression of miR-155 (left) and miR-146a (right) in plasma was determined by TaqMan qRT-real time PCR and spiked C. elegans miR-39 was used as internal control (n=4–6). Fold change over saline treated mice is shown. Data represent mean ± SEM. Non-parametric Mann-Whitney test was employed for statistical analysis.

Fig. 5. Circulating miRNAs localize to the exosome-rich or protein-rich compartments in a liver injury-specific manner

Exosome-rich or protein-rich fractions were isolated with ExoQuick Exosome Precipitation solution (SBI, System Biosciences) from serum or plasma as described in methods and lysed with QIAzole (Qiagen). Total RNA was isolated with miRNeasy kit (Qiagen) and used for miRNA detection. The expression of miR-122 and miR-155 was quantified in exosome-rich or protein-rich fractions isolated from serum (A left & right; ALD model) and plasma (C left & right; APAP model and E left & right; CpG model). B&D&F Expression of miR-122 and miR-155 was quantified in the liver after alcohol (B), APAP (D) and CpG+LPS (F) treatments (n=4–8). Spiked C. elegans miR-39 (serum or plasma) and SnoRNA202 (liver) was used as internal control. The fold change over pair-fed (5A,B) or saline (5C–F) treated mice is shown. Data represent mean ± SEM. * p<0.05 compared to saline treated mice. Non-parametric Mann-Whitney test was employed for statistical analysis.