Skip to main content
SpringerLink
Log in

Neutrophils Play a Crucial Role in the Early Stage of Nonalcoholic Steatohepatitis via Neutrophil Elastase in Mice

  • Original Paper
  • Published:
Cell Biochemistry and Biophysics Aims and scope Submit manuscript

Abstract

Neutrophils infiltration in liver is one of the typical histological characteristics of nonalcoholic steatohepatitis (NASH) in both animal models and human subjects. This study was aimed to investigate the role of neutrophils in the process of NASH and its underling mechanisms. C57BL/6J mice were fed with either standard chow (SC) or methionine/choline-deficient (MCD) diet for 1, 2, 4, 8 weeks, respectively. C57BL/6J APOE−/− mice were fed with SC or high-fat high-cholesterol (HFHC) diet. Anti-Ly6G antibody was employed to deplete neutrophils and sivelestat was used to inhibit neutrophil elastase (NE). MCD-diet-receiving mice with neutrophil depletion had much lower serum ALT activity, liver inflammation, and mRNA levels of proinflammatory genes in the early stage of NASH (1 and 2 weeks) when compared to non-neutrophil-depleted mice. NE inhibitor sivelestat could recapitulate the effects of neutrophil depletion in APOE−/− mice fed with HFHC diet. As the disease progressed (4 and 8 weeks), neutrophil depletion did not lower serum ALT levels and liver lesions due to activation of Kupffer cells. Finally, we found neutrophils also affected anti-inflammation cytokine interleukin-1 receptor antagonist mRNA expression. Neutrophils play a crucial role in the early stage of NASH via NE.

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

Access this article

Log in via an institution

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

Similar content being viewed by others

References

  1. Ong, J. P., Younossi, Z. M. (2007). Epidemiology and natural history of NAFLD and NASH. Clinics in Liver Disease, 11, 1–16, vii.

  2. Xun, Y. H., Fan, J. G., Zang, G. Q., Liu, H., Jiang, Y. M., Xiang, J., et al. (2012). Suboptimal performance of simple noninvasive tests for advanced fibrosis in Chinese patients with nonalcoholic fatty liver disease. Journal of Digestive Diseases, 13, 588–595.

    Article  PubMed  Google Scholar 

  3. Rensen, S. S., Slaats, Y., Nijhuis, J., Jans, A., Bieghs, V., Driessen, A., et al. (2009). Increased hepatic myeloperoxidase activity in obese subjects with nonalcoholic steatohepatitis. American Journal of Pathology, 175, 1473–1482.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Gadd, V. L., Skoien, R., Powell, E. E., Fagan, K. J., Winterford, C., Horsfall, L., et al. (2014). The portal inflammatory infiltrate and ductular reaction in human nonalcoholic fatty liver disease. Hepatology, 59, 1393–1405.

    Article  PubMed  Google Scholar 

  5. Moles, A., Murphy, L., Wilson, C. L., Chakraborty, J. B., Fox, C., Park, E. J., et al. (2014). A TLR2/S100A9/CXCL-2 signaling network is necessary for neutrophil recruitment in acute and chronic liver injury in the mouse. Journal of Hepatology, 60, 782–791.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Alkhouri, N., Morris-Stiff, G., Campbell, C., Lopez, R., Tamimi, T. A., Yerian, L., et al. (2012). Neutrophil to lymphocyte ratio: a new marker for predicting steatohepatitis and fibrosis in patients with nonalcoholic fatty liver disease. Liver International, 32, 297–302.

    Article  CAS  PubMed  Google Scholar 

  7. Daley, J. M., Thomay, A. A., Connolly, M. D., Reichner, J. S., & Albina, J. E. (2008). Use of Ly6G-specific monoclonal antibody to deplete neutrophils in mice. Journal of Leukocyte Biology, 83, 64–70.

    Article  CAS  PubMed  Google Scholar 

  8. Pham, C. T. (2006). Neutrophil serine proteases: specific regulators of inflammation. Nature Reviews Immunology, 6, 541–550.

    Article  CAS  PubMed  Google Scholar 

  9. Talukdar, S., da Oh, Y., Bandyopadhyay, G., Li, D., Xu, J., McNelis, J., et al. (2012). Neutrophils mediate insulin resistance in mice fed a high-fat diet through secreted elastase. Nature Medicine, 18, 1407–1412.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Mansuy-Aubert, V., Zhou, Q. L., Xie, X., Gong, Z., Huang, J. Y., Khan, A. R., et al. (2013). Imbalance between neutrophil elastase and its inhibitor alpha1-antitrypsin in obesity alters insulin sensitivity, inflammation, and energy expenditure. Cell Metabolism, 17, 534–548.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Stankovic, M. N., Mladenovic, D. R., Duricic, I., Sobajic, S. S., Timic, J., Jorgacevic, B., et al. (2014). Time-dependent changes and association between liver free fatty acids, serum lipid profile and histological features in mice model of nonalcoholic fatty liver disease. Archives of Medical Research, 45, 116–124.

    Article  CAS  PubMed  Google Scholar 

  12. Ye, D., Li, F. Y., Lam, K. S., Li, H., Jia, W., Wang, Y., et al. (2012). Toll-like receptor-4 mediates obesity-induced non-alcoholic steatohepatitis through activation of X-box binding protein-1 in mice. Gut, 61, 1058–1067.

    Article  CAS  PubMed  Google Scholar 

  13. Kleiner, D. E., Brunt, E. M., Van Natta, M., Behling, C., Contos, M. J., Cummings, O. W., et al. (2005). Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology, 41, 1313–1321.

    Article  PubMed  Google Scholar 

  14. Liu, J., Zhuang, Z. J., Bian, D. X., Ma, X. J., Xun, Y. H., Yang, W. J., et al. (2014). Toll-like receptor-4 signalling in the progression of non-alcoholic fatty liver disease induced by high-fat and high-fructose diet in mice. Clinical and Experimental Pharmacology and Physiology, 41, 482–488.

    Article  CAS  PubMed  Google Scholar 

  15. Jamieson, T., Clarke, M., Steele, C. W., Samuel, M. S., Neumann, J., Jung, A., et al. (2012). Inhibition of CXCR2 profoundly suppresses inflammation-driven and spontaneous tumorigenesis. Journal of Clinical Investigation, 122, 3127–3144.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Zadelaar, S., Kleemann, R., Verschuren, L., de der Vries-Van Weij, J., van der Hoorn, J., Princen, H. M., et al. (2007). Mouse models for atherosclerosis and pharmaceutical modifiers. Arteriosclerosis, Thrombosis, and Vascular Biology, 27, 1706–1721.

    Article  CAS  PubMed  Google Scholar 

  17. Seki, E., Park, E., & Fujimoto, J. (2011). Toll-like receptor signaling in liver regeneration, fibrosis and carcinogenesis. Hepatology Research, 41, 597–610.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Seki, E., De Minicis, S., Osterreicher, C. H., Kluwe, J., Osawa, Y., Brenner, D. A., et al. (2007). TLR4 enhances TGF-beta signaling and hepatic fibrosis. Nature Medicine, 13, 1324–1332.

    Article  CAS  PubMed  Google Scholar 

  19. Freeman, B. D., & Buchman, T. G. (2001). Interleukin-1 receptor antagonist as therapy for inflammatory disorders. Expert Opinion on Biological Therapy, 1, 301–308.

    Article  CAS  PubMed  Google Scholar 

  20. Kavanaugh, A. (2006). Anakinra (interleukin-1 receptor antagonist) has positive effects on function and quality of life in patients with rheumatoid arthritis. Advances in Therapy, 23, 208–217.

    Article  CAS  PubMed  Google Scholar 

  21. Akash, M. S., Shen, Q., Rehman, K., & Chen, S. (2012). Interleukin-1 receptor antagonist: a new therapy for type 2 diabetes mellitus. Journal of Pharmaceutical Sciences, 101, 1647–1658.

    Article  CAS  PubMed  Google Scholar 

  22. Akash, M. S., Rehman, K., & Chen, S. (2013). An overview of valuable scientific models for diabetes mellitus. Current Diabetes Review, 9, 286–293.

    Article  Google Scholar 

  23. Seckinger, P., Lowenthal, J. W., Williamson, K., Dayer, J. M., & MacDonald, H. R. (1987). A urine inhibitor of interleukin 1 activity that blocks ligand binding. Journal of Immunology, 139, 1546–1549.

    CAS  Google Scholar 

  24. Mocsai, A. (2013). Diverse novel functions of neutrophils in immunity, inflammation, and beyond. Journal of Experimental Medicine, 210, 1283–1299.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Drechsler, M., Megens, R. T., van Zandvoort, M., Weber, C., & Soehnlein, O. (2010). Hyperlipidemia-triggered neutrophilia promotes early atherosclerosis. Circulation, 122, 1837–1845.

    Article  CAS  PubMed  Google Scholar 

  26. Doring, Y., Drechsler, M., Wantha, S., Kemmerich, K., Lievens, D., Vijayan, S., et al. (2012). Lack of neutrophil-derived CRAMP reduces atherosclerosis in mice. Circulation Research, 110, 1052–1056.

    Article  PubMed  Google Scholar 

  27. von Bruhl, M. L., Stark, K., Steinhart, A., Chandraratne, S., Konrad, I., Lorenz, M., et al. (2012). Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo. Journal of Experimental Medicine, 209, 819–835.

    Article  Google Scholar 

  28. Piwowar, A., Knapik-Kordecka, M., & Warwas, M. (2000). Concentration of leukocyte elastase in plasma and polymorphonuclear neutrophil extracts in type 2 diabetes. Clinical Chemistry and Laboratory Medicine, 38, 1257–1261.

    Article  CAS  PubMed  Google Scholar 

  29. Bizbiz, L., Bonithon-Kopp, C., Ducimetiere, P., Berr, C., Alperovitch, A., & Robert, L. (1996). Relation of serum elastase activity to ultrasonographically assessed carotid artery wall lesions and cardiovascular risk factors. The EVA study. Atherosclerosis, 120, 47–55.

    Article  CAS  PubMed  Google Scholar 

  30. Takemasa, A., Ishii, Y., & Fukuda, T. (2012). A neutrophil elastase inhibitor prevents bleomycin-induced pulmonary fibrosis in mice. European Respiratory Journal, 40, 1475–1482.

    Article  CAS  PubMed  Google Scholar 

  31. Fox, J., & Haston, C. K. (2013). CXC receptor 1 and 2 and neutrophil elastase inhibitors alter radiation-induced lung disease in the mouse. International Journal of Radiation Oncology Biology Physics, 85, 215–222.

    Article  CAS  Google Scholar 

  32. Sakashita, A., Nishimura, Y., Nishiuma, T., Takenaka, K., Kobayashi, K., Kotani, Y., et al. (2007). Neutrophil elastase inhibitor (sivelestat) attenuates subsequent ventilator-induced lung injury in mice. European Journal of Pharmacology, 571, 62–71.

    Article  CAS  PubMed  Google Scholar 

  33. Faria, M. S., Calegari-Silva, T. C., de Carvalho Vivarini, A., Mottram, J. C., Lopes, U. G., & Lima, A. P. (2014). Role of protein kinase R in the killing of Leishmania major by macrophages in response to neutrophil elastase and TLR4 via TNFalpha and IFNbeta. FASEB Journal, 28, 3050–3063.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. McDonald, B., Jenne, C. N., Zhuo, L., Kimata, K., & Kubes, P. (2013). Kupffer cells and activation of endothelial TLR4 coordinate neutrophil adhesion within liver sinusoids during endotoxemia. American Journal of Physiology-Gastrointestinal and Liver Physiology, 305, G797–G806.

    Article  CAS  PubMed  Google Scholar 

  35. Serhan, C. N., Chiang, N., & Van Dyke, T. E. (2008). Resolving inflammation: dual anti-inflammatory and pro-resolution lipid mediators. Nature Reviews Immunology, 8, 349–361.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. El Kebir, D., & Filep, J. G. (2010). Role of neutrophil apoptosis in the resolution of inflammation. Scientific World Journal, 10, 1731–1748.

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

This study was supported by Natural Science Foundation of Zhejiang Province (LY14H070004); Science and Technology Development Planning Project of Hangzhou City (20140633B09).

Author information

Authors and Affiliations

  1. Department of Endocrinology, The Affiliated Hospital of Hangzhou Normal University, Hangzhou, Zhejiang, China

    Shufei Zang, Lei Wang, Xiaojie Ma & Geli Zhu

  2. Zhejiang Chinese Medical University, Hangzhou, Zhejiang, China

    Shufei Zang

  3. Center for Translational Medicine, The Affiliated Hospital of Hangzhou Normal University, Hangzhou, Zhejiang, China

    Zhenjie Zhuang, Yan Luo, Yinlan Liu & Junping Shi

  4. Department of Liver Diseases, The Affiliated Hospital of Hangzhou Normal University, 126 Wenzhou Road, Hangzhou, 310015, Zhejiang, China

    Junping Shi

  5. Department of Liver Diseases, Xixi Hospital of Hangzhou, Hangzhou, Zhejiang, China

    Yunhao Xun

  6. Department of Health Management, School of Medicine, Hangzhou Normal University, Hangzhou, Zhejiang, China

    Falin Zhao

  7. Department of Pathology, The Affiliated Hospital of Hangzhou Normal University, Hangzhou, Zhejiang, China

    Wenjun Yang & Juan Liu

  8. State Key Laboratory of Pharmaceutical Biotechnology, Department of Medicine, Faculty of Medicine, The University of Hong Kong, Room L8-33A, Lab Block, 21 Sassoon Road, Hong Kong, China

    Dewei Ye

Authors
  1. Shufei Zang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lei Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xiaojie Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Geli Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zhenjie Zhuang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yunhao Xun
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Falin Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Wenjun Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Juan Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Yan Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Yinlan Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Dewei Ye
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Junping Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Dewei Ye or Junping Shi.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zang, S., Wang, L., Ma, X. et al. Neutrophils Play a Crucial Role in the Early Stage of Nonalcoholic Steatohepatitis via Neutrophil Elastase in Mice. Cell Biochem Biophys 73, 479–487 (2015). https://doi.org/10.1007/s12013-015-0682-9

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12013-015-0682-9

Keywords

Search

Navigation