, Volume 57, Issue 1, pp 72–82 | Cite as

Neutrophil depletion improves diet-induced non-alcoholic fatty liver disease in mice

  • Rongying Ou
  • Jia Liu
  • Mingfen Lv
  • Jingying Wang
  • Jinmeng Wang
  • Li Zhu
  • Liang ZhaoEmail author
  • Yunsheng XuEmail author
Original Article



Non-alcoholic fatty liver disease is highly associated with morbidity and mortality in population. Although studies have already demonstrated that the immune response plays a pivotal role in the development of non-alcoholic fatty liver disease, the comprehensive regulation is unclear. Therefore, present study was carried out to investigate the non-alcoholic fatty liver disease development under neutrophil depletion.


To achieve the aim of the study, C57BL/6 J mice were fed with high fat diet for 6 weeks before treated with neutrophil deplete antibody 1A8 or isotype control (200 μg/ mouse every week) for another 4 weeks.


Treated with 1A8 antibody, obese mice exhibited better whole body metabolic parameters, including reduction of body weight gain and fasting blood glucose levels. Neutrophil depletion also effectively reduced hepatic structural disorders, dysfunction and lipid accumulation. Lipid β-oxidative markers, phosphorylated-AMP-activated protein kinase α and phosphorylated-acetyl-CoA carboxylase levels were increased in 1A8 antibody-treated obese mouse group. The mitochondrial number and function were also reversed after 1A8 antibody treatment, including increased mitochondrial number, reduced lipid oxidative damage and enhanced mitochondrial activity. Furthermore, the expression of inflammatory cytokines, tumor necrosis factor-α, interleukin-6, and monocyte chemoattractant protein-1 were obviously reduced after neutrophil depletion, accompanied with decreased F4/80 mRNA level and macrophage percentage in liver. The decreased NF-κB signaling activity was also involved in the beneficial effect of neutrophil depletion.


Taken together, neutrophil depletion could attenuate metabolic syndromes and hepatic dysfunction.


Neutrophil Non-alcoholic fatty liver disease Lipid β-oxidation Mitochondria Inflammation 



This study was supported by the National Natural Science Foundation of China (81571395, 81373075 and 81371748).


This study was funded by the National Natural Science Foundation of China (81571395, 81373075 and 81371748).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

Procedures involving the animal experiments were approved by the Wenzhou Medical University Animal Policy and Welfare Committee.

Supplementary material

12020_2017_1323_MOESM1_ESM.pdf (159 kb)
Supplementary Information


  1. 1.
    P.L. Jansen, Non-alcoholic steatohepatitis. Eur. J. Gastroenterol. Hepatol. 16, 1079–1085 (2004)CrossRefPubMedGoogle Scholar
  2. 2.
    C.A. Matteoni, Z.M. Younossi, T. Gramlich, N. Boparai, Y.C. Liu, A.J. McCullough, Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity. Gastroenterology 116, 1413–1419 (1999)CrossRefPubMedGoogle Scholar
  3. 3.
    C.P. Day, O.F. James, Steatohepatitis: a tale of two “hits” ? Gastroenterology 114, 842–845 (1998)CrossRefPubMedGoogle Scholar
  4. 4.
    A. Leroux, G. Ferrere, V. Godie, F. Cailleux, M.L. Renoud, F. Gaudin, S. Naveau, S. Prevot, S. Makhzami, G. Perlemuter, A.M. Cassard-Doulcier, Toxic lipids stored by Kupffer cells correlates with their pro-inflammatory phenotype at an early stage of steatohepatitis. J. Hepatol. 57, 141–149 (2012)CrossRefPubMedGoogle Scholar
  5. 5.
    T. Miyake, S.M. Akbar, O. Yoshida, S. Chen, Y. Hiasa, B. Matsuura, M. Abe, M. Onji, Impaired dendritic cell functions disrupt antigen-specific adaptive immune responses in mice with nonalcoholic fatty liver disease. J. Gastroenterol. 45, 859–867 (2010)CrossRefPubMedGoogle Scholar
  6. 6.
    R. Xu, H. Huang, Z. Zhang, F.S. Wang, The role of neutrophils in the development of liver diseases. Cell. Mol. Immunol. 11, 224–231 (2014b)CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    M. Donini, S. Fontana, G. Savoldi, W. Vermi, L. Tassone, F. Gentili, E. Zenaro, D. Ferrari, L.D. Notarangelo, F. Porta, F. Facchetti, L.D. Notarangelo, S. Dusi, R. Badolato, G-CSF treatment of severe congenital neutropenia reverses neutropenia but does not correct the underlying functional deficiency of the neutrophil in defending against microorganisms. Blood 109, 4716–4723 (2007)CrossRefPubMedGoogle Scholar
  8. 8.
    B. McDonald, E.F. McAvoy, F. Lam, V. Gill, C. de la Motte, R.C. Savani, P. Kubes, Interaction of CD44 and hyaluronan is the dominant mechanism for neutrophil sequestration in inflamed liver sinusoids. J. Exp. Med. 205, 915–927 (2008)CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    N. Alkhouri, G. Morris-Stiff, C. Campbell, R. Lopez, T.A. Tamimi, L. Yerian, N.N. Zein, A.E. Feldstein, Neutrophil to lymphocyte ratio: a new marker for predicting steatohepatitis and fibrosis in patients with nonalcoholic fatty liver disease. Liver Int. 32, 297–302 (2012)CrossRefPubMedGoogle Scholar
  10. 10.
    C.Z. Larter, M.M. Yeh, W.G. Haigh, D.M. Van Rooyen, J. Brooling, D. Heydet, C.J. Nolan, N.C. Teoh, G.C. Farrell, Dietary modification dampens liver inflammation and fibrosis in obesity-related fatty liver disease. Obesity 21, 1189–1199 (2013)CrossRefPubMedGoogle Scholar
  11. 11.
    E.J. Park, J.H. Lee, G.Y. Yu, G. He, S.R. Ali, R.G. Holzer, C.H. Osterreicher, H. Takahashi, M. Karin, Dietary and genetic obesity promote liver inflammation and tumorigenesis by enhancing IL-6 and TNF expression. Cell 140, 197–208 (2010)CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    R. Ibusuki, H. Uto, S. Arima, S. Mawatari, Y. Setoguchi, Y. Iwashita, S. Hashimoto, T. Maeda, S. Tanoue, S. Kanmura, M. Oketani, A. Ido, H. Tsubouchi, Transgenic expression of human neutrophil peptide-1 enhances hepatic fibrosis in mice fed a choline-deficient, l-amino acid-defined diet. Liver Int. 33, 1549–1556 (2013)PubMedGoogle Scholar
  13. 13.
    M.P. Murphy, How mitochondria produce reactive oxygen species. Biochem. J. 417, 1–13 (2009)CrossRefPubMedGoogle Scholar
  14. 14.
    C. Koliaki, J. Szendroedi, K. Kaul, T. Jelenik, P. Nowotny, F. Jankowiak, C. Herder, M. Carstensen, M. Krausch, W.T. Knoefel, M. Schlensak, M. Roden, Adaptation of hepatic mitochondrial function in humans with non-alcoholic fatty liver is lost in steatohepatitis. Cell Metab. 21, 739–746 (2015)CrossRefPubMedGoogle Scholar
  15. 15.
    J. Chida, K. Yamane, T. Takei, H. Kido, An efficient extraction method for quantitation of adenosine triphosphate in mammalian tissues and cells. Anal. Chim. Acta 727, 8–12 (2012)CrossRefPubMedGoogle Scholar
  16. 16.
    K.E. Duberley, A.Y. Abramov, A. Chalasani, S.J. Heales, S. Rahman, I.P. Hargreaves, Human neuronal coenzyme Q10 deficiency results in global loss of mitochondrial respiratory chain activity, increased mitochondrial oxidative stress and reversal of ATP synthase activity: implications for pathogenesis and treatment. J. Inherit. Metab. Dis. 36, 63–73 (2013)CrossRefPubMedGoogle Scholar
  17. 17.
    M.A. Selak, J.P. de Chadarevian, J.J. Melvin, W.D. Grover, L. Salganicoff, E.M. Kaye, Mitochondrial activity in Pompe’s disease. Pediatr. Neurol. 23, 54–57 (2000)CrossRefPubMedGoogle Scholar
  18. 18.
    Z.F. Ba, P. Wang, D.J. Koo, W.G. Cioffi, K.I. Bland, I.H. Chaudry, Alterations in tissue oxygen consumption and extraction after trauma and hemorrhagic shock. Crit. Care Med. 28, 2837–2842 (2000)CrossRefPubMedGoogle Scholar
  19. 19.
    G. Hofhaus, R.M. Shakeley, G. Attardi, Use of polarography to detect respiration defects in cell cultures. Methods Enzymol. 264, 476–483 (1996)CrossRefPubMedGoogle Scholar
  20. 20.
    J. Xu, K. Cao, Y. Li, X. Zou, C. Chen, I.M. Szeto, Z. Dong, Y. Zhao, Y. Shi, J. Wang, J. Liu, Z. Feng, Bitter gourd inhibits the development of obesity-associated fatty liver in C57BL/6 mice fed a high-fat diet. J. Nutr. 144, 475–483 (2014a)CrossRefPubMedGoogle Scholar
  21. 21.
    A. Carobene, F. Braga, T. Roraas, S. Sandberg, W.A. Bartlett, A systematic review of data on biological variation for alanine aminotransferase, aspartate aminotransferase and gamma-glutamyl transferase. Clin. Chem. Lab. Med. 51, 1997–2007 (2013)CrossRefPubMedGoogle Scholar
  22. 22.
    A. Herms, M. Bosch, B.J. Reddy, N.L. Schieber, A. Fajardo, C. Ruperez, A. Fernandez-Vidal, C. Ferguson, C. Rentero, F. Tebar, C. Enrich, R.G. Parton, S.P. Gross, A. Pol, AMPK activation promotes lipid droplet dispersion on detyrosinated microtubules to increase mitochondrial fatty acid oxidation. Nat. Commun. 6, 7176 (2015)CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    X. Fu, R.M. Chin, L. Vergnes, H. Hwang, G. Deng, Y. Xing, M.Y. Pai, S. Li, L. Ta, F. Fazlollahi, C. Chen, R.M. Prins, M.A. Teitell, D.A. Nathanson, A. Lai, K.F. Faull, M. Jiang, S.G. Clarke, T.F. Cloughesy, T.G. Graeber, D. Braas, H.R. Christofk, M.E. Jung, K. Reue, J. Huang, 2-Hydroxyglutarate inhibits ATP synthase and mTOR signaling. Cell Metab. 22, 508–515 (2015)CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    K. Miura, L. Yang, N. van Rooijen, H. Ohnishi, E. Seki, Hepatic recruitment of macrophages promotes nonalcoholic steatohepatitis through CCR2. Am. J. Physiol. Gastrointest. Liver Physiol. 302, G1310–G1321 (2012)CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    V. Bieghs, P.C. Rensen, M.H. Hofker, R. Shiri-Sverdlov, NASH and atherosclerosis are two aspects of a shared disease: central role for macrophages. Atherosclerosis 220, 287–293 (2012)CrossRefPubMedGoogle Scholar
  26. 26.
    K.C. McGrath, X.H. Li, P.T. Whitworth, R. Kasz, J.T. Tan, S.V. McLennan, D.S. Celermajer, P.J. Barter, K.A. Rye, A.K. Heather, High density lipoproteins improve insulin sensitivity in high-fat diet-fed mice by suppressing hepatic inflammation. J. Lipid Res. 55, 421–430 (2014)CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    E. Passos, C.D. Pereira, I.O. Goncalves, S. Rocha-Rodrigues, N. Silva, J.T. Guimaraes, D. Neves, A. Ascensao, J. Magalhaes, M.J. Martins, Role of physical exercise on hepatic insulin, glucocorticoid and inflammatory signaling pathways in an animal model of non-alcoholic steatohepatitis. Life Sci. 123, 51–60 (2015)CrossRefPubMedGoogle Scholar
  28. 28.
    P. Filipazzi, R. Valenti, V. Huber, L. Pilla, P. Canese, M. Iero, C. Castelli, L. Mariani, G. Parmiani, L. Rivoltini, Identification of a new subset of myeloid suppressor cells in peripheral blood of melanoma patients with modulation by a granulocyte-macrophage colony-stimulation factor-based antitumor vaccine. J. Clin. Oncol. 25, 2546–2553 (2007)CrossRefPubMedGoogle Scholar
  29. 29.
    K. Wing, S. Sakaguchi, Regulatory T cells exert checks and balances on self tolerance and autoimmunity. Nat. Immunol. 11, 7–13 (2010)CrossRefPubMedGoogle Scholar
  30. 30.
    R. Bottino, L.A. Fernandez, C. Ricordi, R. Lehmann, M.F. Tsan, R. Oliver, L. Inverardi, Transplantation of allogeneic islets of Langerhans in the rat liver: effects of macrophage depletion on graft survival and microenvironment activation. Diabetes 47, 316–323 (1998)CrossRefPubMedGoogle Scholar
  31. 31.
    R.I. Tepper, R.L. Coffman, P. Leder, An eosinophil-dependent mechanism for the antitumor effect of interleukin-4. Science 257, 548–551 (1992)CrossRefPubMedGoogle Scholar
  32. 32.
    J.M. Daley, A.A. Thomay, M.D. Connolly, J.S. Reichner, J.E. Albina, Use of Ly6G-specific monoclonal antibody to deplete neutrophils in mice. J. Leukoc. Biol. 83, 64–70 (2008)CrossRefPubMedGoogle Scholar
  33. 33.
    D.E. Dulek, D.C. Newcomb, K. Goleniewska, J. Cephus, W. Zhou, S. Reiss, S. Toki, F. Ye, R. Zaynagetdinov, T.P. Sherrill, T.S. Blackwell, M.L. Moore, K.L. Boyd, J.K. Kolls, R.S. Peebles Jr., Allergic airway inflammation decreases lung bacterial burden following acute Klebsiella pneumoniae infection in a neutrophil- and CCL8-dependent manner. Infect. Immun. 82, 3723–3739 (2014)CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Z.G. Fridlender, J. Sun, S. Kim, V. Kapoor, G. Cheng, L. Ling, G.S. Worthen, S.M. Albelda, Polarization of tumor-associated neutrophil phenotype by TGF-beta: “N1” versus “N2” TAN. Cancer Cell 16, 183–194 (2009)CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    C.D. Williams, M.L. Bajt, M.R. Sharpe, M.R. McGill, A. Farhood, H. Jaeschke, Neutrophil activation during acetaminophen hepatotoxicity and repair in mice and humans. Toxicol. Appl. Pharmacol. 275, 122–133 (2014)CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    A.P. de Vries, P. Ruggenenti, X.Z. Ruan, M. Praga, J.M. Cruzado, I.M. Bajema, V.D. D’Agati, H.J. Lamb, D. Pongrac Barlovic, R. Hojs, M. Abbate, R. Rodriquez, C.E. Mogensen, E. Porrini; Diabesity E-EWG, Fatty kidney: emerging role of ectopic lipid in obesity-related renal disease. Lancet Diabetes Endocrinol. 2, 417–426 (2014)CrossRefPubMedGoogle Scholar
  37. 37.
    L. Martinez-Rubio, S. Morais, O. Evensen, S. Wadsworth, J.G. Vecino, K. Ruohonen, J.G. Bell, D.R. Tocher, Effect of functional feeds on fatty acid and eicosanoid metabolism in liver and head kidney of Atlantic salmon (Salmo salar L.) with experimentally induced heart and skeletal muscle inflammation. Fish Shellfish. Immunol. 34, 1533–1545 (2013)CrossRefPubMedGoogle Scholar
  38. 38.
    A.W. Bell, Lipid metabolism in liver and selected tissues and in the whole body of ruminant animals. Prog. Lipid Res. 18, 117–164 (1979)CrossRefPubMedGoogle Scholar
  39. 39.
    W. Otten, C. Wirth, P.A. Iaizzo, H.M. Eichinger, A high omega 3 fatty acid diet alters fatty acid composition of heart, liver, kidney, adipose tissue and skeletal muscle in swine. Ann. Nutr. Metab. 37, 134–141 (1993)CrossRefPubMedGoogle Scholar
  40. 40.
    F. Nassir, J.A. Ibdah, Role of mitochondria in nonalcoholic fatty liver disease. Int. J. Mol. Sci. 15, 8713–8742 (2014)CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    A.B. Santamarina, M. Carvalho-Silva, L.M. Gomes, M.H. Okuda, A.A. Santana, E.L. Streck, M. Seelaender, C.M. Oller do Nascimento, E.B. Ribeiro, F.S. Lira, L.M. Oyama, Decaffeinated green tea extract rich in epigallocatechin-3-gallate prevents fatty liver disease by increased activities of mitochondrial respiratory chain complexes in diet-induced obesity mice. J. Nutr. Biochem 26, 1348–1356 (2015)CrossRefPubMedGoogle Scholar
  42. 42.
    J. Ye, Mechanisms of insulin resistance in obesity. Front. Med. 7, 14–24 (2013)CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    S.S. Rensen, V. Bieghs, S. Xanthoulea, E. Arfianti, J.A. Bakker, R. Shiri-Sverdlov, M.H. Hofker, J.W. Greve, W.A. Buurman, Neutrophil-derived myeloperoxidase aggravates non-alcoholic steatohepatitis in low-density lipoprotein receptor-deficient mice. PLoS One 7, e52411 (2012)CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    S. Talukdar, Y. Oh da, G. Bandyopadhyay, D. Li, J. Xu, J. McNelis, M. Lu, P. Li, Q. Yan, Y. Zhu, J. Ofrecio, M. Lin, M.B. Brenner, J.M. Olefsky, Neutrophils mediate insulin resistance in mice fed a high-fat diet through secreted elastase. Nat. Med. 18, 1407–1412 (2012)CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Rongying Ou
    • 1
    • 2
  • Jia Liu
    • 1
    • 3
  • Mingfen Lv
    • 1
    • 3
  • Jingying Wang
    • 1
    • 3
  • Jinmeng Wang
    • 1
    • 3
  • Li Zhu
    • 1
    • 3
  • Liang Zhao
    • 1
    • 4
    Email author
  • Yunsheng Xu
    • 1
    • 3
    Email author
  1. 1.Laboratory for Advanced Interdisciplinary Research, Institutes of Translational MedicineThe First Affiliated Hospital of Wenzhou Medical UniversityWenzhouChina
  2. 2.Department of Gynaecology and ObstetricsThe First Affiliated Hospital of Wenzhou Medical UniversityWenzhouChina
  3. 3.Department of DermatovenereologyThe First Affiliated Hospital of Wenzhou Medical UniversityWenzhouChina
  4. 4.Division of PET/CT, Department of RadiologyThe First Affiliated Hospital of Wenzhou Medical UniversityWenzhouChina

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