[go: up one dir, main page]
Skip to main content

Advertisement

Springer Nature Link
Log in

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

  • Original Article
  • Published:
Journal of Molecular Medicine Aims and scope Submit manuscript

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

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

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

References

  1. Yin M, Wheeler MD, Kono H, Bradford BU, Gallucci RM, Luster MI, Thurman RG (1999) Essential role of tumor necrosis factor alpha in alcohol-induced liver injury in mice. Gastroenterology 117:942–952

    Article  CAS  Google Scholar 

  2. Mathurin P (2009) Alcohol and the liver. Gastroenterologie clinique et biologique 33:840–849

    Article  CAS  Google Scholar 

  3. Nanji AA, Su GL, Laposata M, French SW (2002) Pathogenesis of alcoholic liver disease--recent advances. Alcohol Clin Exp Res 26:731–736

    PubMed  Google Scholar 

  4. Feldstein AE, Gores GJ (2005) Apoptosis in alcoholic and nonalcoholic steatohepatitis. Frontiers in bioscience : a journal and virtual library 10:3093–3099

    Article  CAS  Google Scholar 

  5. Thurman RG, Bradford BU, Iimuro Y, Knecht KT, Connor HD, Adachi Y, Wall C, Arteel GE, Raleigh JA, Forman DT, Mason RP (1997) Role of Kupffer cells, endotoxin and free radicals in hepatotoxicity due to prolonged alcohol consumption: studies in female and male rats. J Nutr 127:903S–906S

    Article  CAS  Google Scholar 

  6. Nanji AA (2002) Role of Kupffer cells in alcoholic hepatitis. Alcohol 27:13–15

    Article  CAS  Google Scholar 

  7. Rose ML, Rusyn I, Bojes HK, Belyea J, Cattley RC, Thurman RG (2000) Role of Kupffer cells and oxidants in signaling peroxisome proliferator-induced hepatocyte proliferation. Mutat Res 448:179–192

    Article  CAS  Google Scholar 

  8. Eguchi A, Lazaro RG, Wang J, Kim J, Povero D, Willliams B, Ho SB, Starkel P, Schnabl B, Ohno-Machado L et al (2017) Extracellular vesicles released by hepatocytes from gastric infusion model of alcoholic liver disease contain a MicroRNA barcode that can be detected in blood. Hepatology 65:475–490

    Article  CAS  Google Scholar 

  9. Lazaro R, Wu R, Lee S, Zhu NL, Chen CL, French SW, Xu J, Machida K, Tsukamoto H (2015) Osteopontin deficiency does not prevent but promotes alcoholic neutrophilic hepatitis in mice. Hepatology 61:129–140

    Article  CAS  Google Scholar 

  10. Yanez-Mo M, Siljander PR, Andreu Z, Zavec AB, Borras FE, Buzas EI, Buzas K, Casal E, Cappello F, Carvalho J et al (2015) Biological properties of extracellular vesicles and their physiological functions. Journal of extracellular vesicles 4:27066

    Article  Google Scholar 

  11. Gao B, Ahmad MF, Nagy LE, Tsukamoto H (2019) Inflammatory pathways in alcoholic steatohepatitis. J Hepatol 70:249–259

    Article  Google Scholar 

  12. Khanova E, Wu R, Wang W, Yan R, Chen Y, French SW, Llorente C, Pan SQ, Yang Q, Li Y, Lazaro R, Ansong C, Smith RD, Bataller R, Morgan T, Schnabl B, Tsukamoto H (2018) Pyroptosis by caspase11/4-gasdermin-D pathway in alcoholic hepatitis in mice and patients. Hepatology 67:1737–1753

    Article  CAS  Google Scholar 

  13. Lai KKY, Kweon SM, Chi F, Hwang E, Kabe Y, Higashiyama R, Qin L, Yan R, Wu RP, Lai K, Fujii N, French S, Xu J, Wang JY, Murali R, Mishra L, Lee JS, Ntambi JM, Tsukamoto H (2017) Stearoyl-CoA desaturase promotes liver fibrosis and tumor development in mice via a Wnt positive-signaling loop by stabilization of low-density lipoprotein-receptor-related proteins 5 and 6. Gastroenterology 152:1477–1491

    Article  CAS  Google Scholar 

  14. Xu J, Chi F, Guo T, Punj V, Lee WN, French SW, Tsukamoto H (2015) NOTCH reprograms mitochondrial metabolism for proinflammatory macrophage activation. J Clin Invest 125:1579–1590

    Article  Google Scholar 

  15. Anders S, Huber W (2010) Differential expression analysis for sequence count data. Genome Biol 11:R106

    Article  CAS  Google Scholar 

  16. Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J Roy Stat Soc B 57:289–300

    Google Scholar 

  17. Kim J, Levy E, Ferbrache A, Stepanowsky P, Farcas C, Wang S, Brunner S, Bath T, Wu Y, Ohno-Machado L (2014) MAGI: a Node.js web service for fast microRNA-Seq analysis in a GPU infrastructure. Bioinformatics 30:2826–2827

    Article  CAS  Google Scholar 

  18. Vlachos IS, Zagganas K, Paraskevopoulou MD, Georgakilas G, Karagkouni D, Vergoulis T, Dalamagas T, Hatzigeorgiou AG (2015) DIANA-miRPath v3.0: deciphering microRNA function with experimental support. Nucleic Acids Res 43:W460–W466

    Article  CAS  Google Scholar 

  19. Wang H, Qian WJ, Mottaz HM, Clauss TR, Anderson DJ, Moore RJ, Camp DG 2nd, Khan AH, Sforza DM, Pallavicini M et al (2005) Development and evaluation of a micro- and nanoscale proteomic sample preparation method. J Proteome Res 4:2397–2403

    Article  CAS  Google Scholar 

  20. Yang J, Yin L, Lessner FH, Nakayasu ES, Payne SH, Fixen KR, Gallagher L, Harwood CS (2017) Genes essential for phototrophic growth by a purple alphaproteobacterium. Environ Microbiol 19:3567–3578

    Article  CAS  Google Scholar 

  21. Bantscheff M, Schirle M, Sweetman G, Rick J, Kuster B (2007) Quantitative mass spectrometry in proteomics: a critical review. Anal Bioanal Chem 389:1017–1031

    Article  CAS  Google Scholar 

  22. Schulze WX, Usadel B (2010) Quantitation in mass-spectrometry-based proteomics. Annu Rev Plant Biol 61:491–516

    Article  CAS  Google Scholar 

  23. Garcia-Martinez I, Santoro N, Chen Y, Hoque R, Ouyang X, Caprio S, Shlomchik MJ, Coffman RL, Candia A, Mehal WZ (2016) Hepatocyte mitochondrial DNA drives nonalcoholic steatohepatitis by activation of TLR9. J Clin Invest 126:859–864

    Article  Google Scholar 

  24. Tkach M, Thery C (2016) Communication by extracellular vesicles: where we are and where we need to go. Cell 164:1226–1232

    Article  CAS  Google Scholar 

  25. Liu X, Rosenthal SB, Meshgin N, Baglieri J, Musallam SG, Diggle K, Lam K, Wu R, Pan SQ, Chen Y, Dorko K, Presnell S, Benner C, Hosseini M, Tsukamoto H, Brenner D, Kisseleva T (2020) Primary alcohol-activated human and mouse hepatic stellate cells share similarities in gene-expression profiles. Hepatology communications 4:606–626

    Article  CAS  Google Scholar 

  26. Liu X, Xu J, Brenner DA, Kisseleva T (2013) Reversibility of liver fibrosis and inactivation of fibrogenic myofibroblasts. Curr Pathobiol Rep 1:209–214

    Article  Google Scholar 

  27. Hirsova P, Ibrahim SH, Verma VK, Morton LA, Shah VH, LaRusso NF, Gores GJ, Malhi H (2016) Extracellular vesicles in liver pathobiology: small particles with big impact. Hepatology 64:2219–2233

    Article  Google Scholar 

  28. Povero D, Panera N, Eguchi A, Johnson CD, Papouchado BG, de Araujo HL, Pinatel EM, Alisi A, Nobili V, Feldstein AE (2015) Lipid-induced hepatocyte-derived extracellular vesicles regulate hepatic stellate cell via microRNAs targeting PPAR-gamma. Cellular and molecular gastroenterology and hepatology 1:646–663 e644

    Article  Google Scholar 

  29. Meng F, Wang K, Aoyama T, Grivennikov SI, Paik Y, Scholten D, Cong M, Iwaisako K, Liu X, Zhang M, Österreicher CH, Stickel F, Ley K, Brenner DA, Kisseleva T (2012) Interleukin-17 signaling in inflammatory, Kupffer cells, and hepatic stellate cells exacerbates liver fibrosis in mice. Gastroenterology 143:765–776 e763

    Article  CAS  Google Scholar 

  30. Fabre T, Kared H, Friedman SL, Shoukry NH (2014) IL-17A enhances the expression of profibrotic genes through upregulation of the TGF-beta receptor on hepatic stellate cells in a JNK-dependent manner. J Immunol 193:3925–3933

    Article  CAS  Google Scholar 

  31. Bhan U, Ballinger MN, Zeng X, Newstead MJ, Cornicelli MD, Standiford TJ (2010) Cooperative interactions between TLR4 and TLR9 regulate interleukin 23 and 17 production in a murine model of gram negative bacterial pneumonia. PLoS One 5:e9896

    Article  Google Scholar 

  32. Salloum N, Hussein HM, Jammaz R, Jiche S, Uthman IW, Abdelnoor AM, Rahal EA (2018) Epstein-Barr virus DNA modulates regulatory T-cell programming in addition to enhancing interleukin-17A production via Toll-like receptor 9. PLoS One 13:e0200546

    Article  Google Scholar 

  33. Mansouri A, Gattolliat CH, Asselah T (2018) Mitochondrial dysfunction and signaling in chronic liver diseases. Gastroenterology 155:629–647

    Article  CAS  Google Scholar 

  34. Hoek JB, Cahill A, Pastorino JG (2002) Alcohol and mitochondria: a dysfunctional relationship. Gastroenterology 122:2049–2063

    Article  CAS  Google Scholar 

  35. Szabo G, Bala S (2013) MicroRNAs in liver disease. Nat Rev Gastroenterol Hepatol 10:542–552

    Article  CAS  Google Scholar 

  36. Kitano M, Bloomston PM (2016) Hepatic stellate cells and microRNAs in pathogenesis of liver fibrosis. J Clin Med 5. https://doi.org/10.3390/jcm5030038

  37. MR MG, Staggs VS, Sharpe MR, Lee WM, Jaeschke H, Acute Liver Failure Study G (2014) Serum mitochondrial biomarkers and damage-associated molecular patterns are higher in acetaminophen overdose patients with poor outcome. Hepatology 60:1336–1345

    Google Scholar 

  38. He Y, Feng D, Li M, Gao Y, Ramirez T, Cao H, Kim SJ, Yang Y, Cai Y, Ju C, Wang H, Li J, Gao B (2017) Hepatic mitochondrial DNA/Toll-like receptor 9/MicroRNA-223 forms a negative feedback loop to limit neutrophil overactivation and acetaminophen hepatotoxicity in mice. Hepatology 66:220–234

    Article  CAS  Google Scholar 

  39. Eguchi A, Kostallari E, Feldstein AE, Shah VH (2019) Extracellular vesicles, the liquid biopsy of the future. J Hepatol 70:1292–1294

    Article  Google Scholar 

  40. Eguchi A, Feldstein AE (2018) Extracellular vesicles in non-alcoholic and alcoholic fatty liver diseases. Liver Res 2:30–34

    Article  Google Scholar 

Download references

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.

Author information

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

Authors
  1. Akiko Eguchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rui Yan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Stephanie Q Pan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Raymond Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jihoon Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yibu Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Charles Ansong
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Richard D. Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Mina Tempaku
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Lucila Ohno-Machado
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Yoshiyuki Takei
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Ariel E. Feldstein
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Hidekazu Tsukamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Akiko Eguchi or Hidekazu Tsukamoto.

Ethics declarations

The use and care of the animals was reviewed and approved by the Institutional Animal Care and Use Committee at the University of Southern California.

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

ESM 1

(PDF 819 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

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

Keywords