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Comprehensive characterization of hepatocyte-derived extracellular vesicles identifies direct miRNA-based regulation of hepatic stellate cells and DAMP-based hepatic macrophage IL-1β and IL-17 upregulation in alcoholic hepatitis mice

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Abstract

Extracellular vesicles (EVs) have been growingly recognized as biomarkers and mediators of alcoholic liver disease (ALD) in human and mice. Here we characterized hepatocyte-derived EVs (HC-EVs) and their cargo for their biological functions in a novel murine model that closely resembles liver pathology observed in patients with alcoholic hepatitis (AH), the most severe spectrum of ALD. The numbers of circulating EVs and HC-EVs were significantly increased by 10-fold in AH mice compared with control mice. The miRNA (miR)–seq analysis detected 20 upregulated and 4 downregulated miRNAs (P < 0.001–0.05) in AH-HC-EVs. Treatment of murine primary hepatic stellate cells (HSCs) with AH-HC-EVs induced α-SMA (P < 0.05) and Col1a1 (P < 0.001). Smad7 and Nr1d2 genes, which were downregulated in HSCs from the AH mice, were predicted targets of 20 miRs upregulated in AH-HC-EVs. Among them were miR-27a and miR-181 which upon transfection in HSCs, indeed repressed Nr1d2, the quiescent HSC marker. AH-HC-EVs were also enriched with organelle proteins and mitochondrial DNA (10-fold, P < 0.05) and upregulated IL-1β and IL-17 production by hepatic macrophages (HMs) from AH mice in a TLR9-dependent manner. These results demonstrate HC-EV release is intensified in AH and suggest that AH-HC-EVs orchestrate liver fibrogenesis by directly targeting the quiescent HSC transcripts via a unique set of miRNAs and by amplifying HSC activation via DAMP-based induction of profibrogenic IL-1β and IL-17 by HMs.

Key messages

• Circulating EVs and HC-EVs were increased in AH mice compared with control mice

• AH-HC-EVs were enriched in miRNAs, organelle proteins, and mitochondrial DNA

• AH-HC-EVs increased cytokine production by AH-HMs in a TLR9-dependent manner

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Abbreviations

ALD:

Alcoholic liver disease

ASH:

Alcoholic steatohepatitis

HCC:

Hepatocellular carcinoma

EV:

Extracellular vesicle

HC:

Hepatocyte

HM:

Hepatic macrophage

HC-EV:

Hepatocyte-derived EV

HM-EV:

Hepatic macrophages-derived EV

miRNA:

MicroRNA

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Acknowledgments

We thank the UCSD IGM Genomics Center, especially Kristen Jepsen for siRNA sequencing advice and assistance and Rui Yan for mtDNA and TLR9-reporter assays. The authors would like to thank the UCSD/CMM electron microscopy facility, especially Timothy Meerloo, for TEM sample preparation and imaging. This EM facility is supported by NIH equipment grant 1S10OD023527. Proteomics work was performed in W. R. Wiley Environmental Molecular Sciences Laboratory (EMSL), a Department of Energy (DOE) office of Biological and Environmental Research (BER) national user facility located at Pacific Northwest National Laboratory (PNNL).

Funding

The work was supported by NIH grants P50AA011999 (Pilot Project Program), R21 AA023574, and JSPS KAKENHI Grant Number JP16H06872 to AE; JSPS KAKENHI Grant Number JP17K09419 to YT; NIH grants U54HL108460 to JK and LOM; NIH grant P41 GM103493 to RDS; NIH grants R01 AA022489 and DK082451 to AEF; NIH grants P50AA011999 (Administrative and Animal Cores), R24AA012885 (Integrative Liver Cell Core), R01AA018663, U01AA027681 and VA grants I01BX001991 (VA Merit Review) and IK6BX004205 (Senior Research Career Scientist award) to HT.

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Authors and Affiliations

  1. Department of Gastroenterology and Hepatology, Graduate School of Medicine, Mie University, Tsu, Japan

    Akiko Eguchi, Mina Tempaku & Yoshiyuki Takei

  2. Department of Pediatrics, University of California San Diego, La Jolla, CA, USA

    Akiko Eguchi & Ariel E. Feldstein

  3. Southern California Research Center for ALPD and Cirrhosis, Los Angeles, CA, USA

    Akiko Eguchi, Rui Yan, Stephanie Q Pan, Raymond Wu, Ariel E. Feldstein & Hidekazu Tsukamoto

  4. JST, PRETO, 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan

    Akiko Eguchi

  5. Department of Pathology, Keck School of Medicine of the University of Southern California, 1333 San Pablo Street, MMR-402, Los Angeles, CA, 90033, USA

    Rui Yan, Stephanie Q Pan, Raymond Wu & Hidekazu Tsukamoto

  6. Department of Biomedical Informatics, University of California San Diego, La Jolla, CA, USA

    Jihoon Kim & Lucila Ohno-Machado

  7. Bioinformatics Services, Keck School of Medicine of the University of Southern California, Los Angeles, CA, 90007, USA

    Yibu Chen

  8. Pacific Northwest National Laboratory, Richland, WA, USA

    Charles Ansong & Richard D. Smith

  9. Greater Los Angeles VA Healthcare System, Los Angeles, CA, USA

    Hidekazu Tsukamoto

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  1. Akiko Eguchi
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Correspondence to Akiko Eguchi or Hidekazu Tsukamoto.

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Eguchi, A., Yan, R., Pan, S.Q. et al. Comprehensive characterization of hepatocyte-derived extracellular vesicles identifies direct miRNA-based regulation of hepatic stellate cells and DAMP-based hepatic macrophage IL-1β and IL-17 upregulation in alcoholic hepatitis mice. J Mol Med 98, 1021–1034 (2020). https://doi.org/10.1007/s00109-020-01926-7

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  • DOI: https://doi.org/10.1007/s00109-020-01926-7

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