NLRP3 deficiency did not attenuate NASH development under high fat calorie diet plus high fructose and glucose in drinking water

Abstract

NOD-like receptor protein 3 (NLRP3) promotes the inflammatory response during progression of nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH). This study aimed to further delineate the role of NLRP3 in NASH development by abolishing its expression in mice. A high-fat and calorie diet plus high fructose and glucose in drinking water (HFCD-HF/G) was used to establish NASH in both wild-type (WT) and NLRP3 knock-out (KO) mice. Hepatocellular injury, hepatic steatosis and fibrosis, as well as inflammatory response and insulin resistance in the liver and epidydimal white adipose tissue (eWAT) were determined. Elevated body weight, liver weight and serum alanine transaminase level, increased hepatic triglyceride accumulation and collagen deposition, and worsened systemic insulin resistance were observed in Nlrp3−/− mice compared to WT mice under HFCD-HF/G feeding. Upregulated hepatic transcription of tumor necrosis factor-α (TNF-α) and monocyte chemotactic protein-1 (MCP-1), and enhanced infiltration of inducible nitric oxide synthase-positive (iNOS+) M1 macrophages were also documented in HFCD-HF/G-fed Nlrp3−/− mice in comparison to HFCD-HF/G-fed WT mice. Moreover, transcription of TNF-α and MCP-1 and infiltration of iNOS+ M1 macrophages were increased in the liver of Nlrp3−/− mice under control diet. NLRP3 deficiency did not attenuate, but instead aggravated NASH development under HFCD-HF/G feeding. The worsened extent of NASH might be attributed to enhanced hepatic MCP-1 expression and M1 macrophage infiltration in Nlrp3−/− mice. Our study points to additional caution when NLRP3 blockade is considered as a therapeutic strategy in the treatment of human NASH.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Genotyping and NLRP3 expression in different mouse groups.
Fig. 2: NLRP3 deficiency aggravated body and liver fat deposition and hepatic injury under HFCD-HF/G feeding.
Fig. 3: NLRP3 deficiency exacerbated hepatic steatosis and collagen deposition under HFCD-HF/G feeding.
Fig. 4: NLRP3 deficiency worsened systemic insulin resistance under HFCD-HF/G feeding.
Fig. 5: Transcription levels of TNF-α, MCP-1 and CD36 expression in the liver.
Fig. 6: Enhanced hepatic infiltration of iNOS+ M1 macrophages with NLRP3 deficiency.

References

  1. 1.

    Diehl AM, Day C. Cause, pathogenesis, and treatment of nonalcoholic steatohepatitis. N Engl J Med. 2017;377:2063–72.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  2. 2.

    Younossi ZM, Koenig AB, Abdelatif D, Fazel Y, Henry L, Wymer M. Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes. Hepatology. 2016;64:73–84.

    PubMed  Article  PubMed Central  Google Scholar 

  3. 3.

    Wu J. Utilization of animal models to investigate nonalcoholic steatohepatitis-associated hepatocellular carcinoma. Oncotarget. 2016;7:42762–76.

    PubMed  PubMed Central  Article  Google Scholar 

  4. 4.

    Ye J, Li TS, Xu G, Zhao YM, Zhang NP, Fan J, et al. JCAD promotes progression of nonalcoholic steatohepatitis to liver cancer by inhibiting LATS2 kinase activity. Cancer Res. 2017;77:5287–5300.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  5. 5.

    Younossi ZM, Otgonsuren M, Henry L, Venkatesan C, Mishra A, Erario M, et al. Association of nonalcoholic fatty liver disease (NAFLD) with hepatocellular carcinoma (HCC) in the United States from 2004 to 2009. Hepatology. 2015;62:1723–30.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  6. 6.

    Kleiner DE, Brunt EM, Wilson LA, Behling C, Guy C, Contos M, et al. Association of histologic disease activity with progression of nonalcoholic fatty liver disease. JAMA Netw Open. 2019;2:e1912565.

    PubMed  PubMed Central  Article  Google Scholar 

  7. 7.

    Tilg H, Moschen AR. Evolution of inflammation in nonalcoholic fatty liver disease: the multiple parallel hits hypothesis. Hepatology. 2010;52:1836–46.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  8. 8.

    Martinon F, Burns K, Tschopp J. The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Mol Cell. 2002;10:417–26.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  9. 9.

    Schroder K, Tschopp J. The inflammasomes. Cell. 2010;140:821–32.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  10. 10.

    Szabo G, Csak T. Inflammasomes in liver diseases. J Hepatol. 2012;57:642–54.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  11. 11.

    Thomas HNAFLD. A critical role for the NLRP3 inflammasome in NASH. Nat Rev Gastroenterol Hepatol. 2017;14:197.

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Wree A, Eguchi A, McGeough MD, Pena CA, Johnson CD, Canbay A, et al. NLRP3 inflammasome activation results in hepatocyte pyroptosis, liver inflammation, and fibrosis in mice. Hepatology. 2014;59:898–910.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. 13.

    Zhang NP, Liu XJ, Xie L, Shen XZ, Wu J. Impaired mitophagy triggers NLRP3 inflammasome activation during the progression from nonalcoholic fatty liver to nonalcoholic steatohepatitis. Lab Invest. 2019;99:749–63.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  14. 14.

    Gaul S, Leszczynska A, Alegre F, Kaufmann B, Johnson CD, Adams LA, et al. Hepatocyte pyroptosis and release of inflammasome particles induce stellate cell activation and liver fibrosis. J Hepatol. 2021;74:156–67.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  15. 15.

    Duan NN, Liu XJ, Wu J. Palmitic acid elicits hepatic stellate cell activation through inflammasomes and hedgehog signaling. Life Sci. 2017;176:42–53.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  16. 16.

    Mridha AR, Wree A, Robertson AAB, Yeh MM, Johnson CD, Van Rooyen DM, et al. NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice. J Hepatol. 2017;66:1037–46.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. 17.

    Henao-Mejia J, Elinav E, Jin C, Hao L, Mehal WZ, Strowig T, et al. Inflammasome-mediated dysbiosis regulates progression of NAFLD and obesity. Nature. 2012;482:179–85.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. 18.

    Pierantonelli I, Rychlicki C, Agostinelli L, Giordano DM, Gaggini M, Fraumene C, et al. Lack of NLRP3-inflammasome leads to gut-liver axis derangement, gut dysbiosis and a worsened phenotype in a mouse model of NAFLD. Sci Rep. 2017;7:12200.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  19. 19.

    Ringling RE, Gastecki ML, Woodford ML, Lum-Naihe KJ, Grant RW, Pulakat L, et al. Loss of Nlrp3 does not protect mice from western diet-induced adipose tissue inflammation and glucose intolerance. PLoS One. 2016;11:e0161939.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  20. 20.

    Liu XJ, Duan NN, Liu C, Niu C, Liu XP, Wu J. Characterization of a murine nonalcoholic steatohepatitis model induced by high fat high calorie diet plus fructose and glucose in drinking water. Lab Invest. 2018;98:1184–99.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  21. 21.

    Wree A, Kahraman A, Gerken G, Canbay A. Obesity affects the liver - the link between adipocytes and hepatocytes. Digestion. 2011;83:124–33.

    PubMed  Article  PubMed Central  Google Scholar 

  22. 22.

    Caputo T, Gilardi F, Desvergne B. From chronic overnutrition to metaflammation and insulin resistance: adipose tissue and liver contributions. FEBS Lett. 2017;591:3061–88.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  23. 23.

    Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology. 2005;41:1313–21.

    PubMed  PubMed Central  Article  Google Scholar 

  24. 24.

    Liu X-J, Xie L, Du K, Liu C, Zhang N-P, Gu C-J, et al. Succinate-GPR-91 receptor signalling is responsible for nonalcoholic steatohepatitis-associated fibrosis: Effects of DHA supplementation. Liver Int. 2020;40:830–43.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  25. 25.

    Ino H. Antigen retrieval by heating en bloc for pre-fixed frozen material. J Histochem Cytochem. 2003;51:995–1003.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  26. 26.

    Yamashita S, Okada Y. Application of heat-induced antigen retrieval to aldehyde-fixed fresh frozen sections. J Histochem Cytochem. 2005;53:1421–32.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  27. 27.

    Manning BD, Toker A. AKT/PKB signaling: navigating the network. Cell. 2017;169:381–405.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. 28.

    Morinaga H, Mayoral R, Heinrichsdorff J, Osborn O, Franck N, Hah N, et al. Characterization of distinct subpopulations of hepatic macrophages in HFD/obese mice. Diabetes. 2015;64:1120–30.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  29. 29.

    Krenkel O, Tacke F. Liver macrophages in tissue homeostasis and disease. Nat Rev Immunol. 2017;17:306–21.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  30. 30.

    Kazankov K, Jorgensen SMD, Thomsen KL, Moller HJ, Vilstrup H, George J, et al. The role of macrophages in nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. Nat Rev Gastroenterol Hepatol. 2019;16:145–59.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  31. 31.

    Krenkel O, Puengel T, Govaere O, Abdallah AT, Mossanen JC, Kohlhepp M, et al. Therapeutic inhibition of inflammatory monocyte recruitment reduces steatohepatitis and liver fibrosis. Hepatology. 2018;67:1270–83.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  32. 32.

    Prochnicki T, Latz E. Inflammasomes on the crossroads of innate immune recognition and metabolic control. Cell Metab. 2017;26:71–93.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  33. 33.

    Lee HM, Kim JJ, Kim HJ, Shong M, Ku BJ, Jo EK. Upregulated NLRP3 inflammasome activation in patients with type 2 diabetes. Diabetes. 2013;62:194–204.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  34. 34.

    Lech M, Lorenz G, Kulkarni OP, Grosser MO, Stigrot N, Darisipudi MN, et al. NLRP3 and ASC suppress lupus-like autoimmunity by driving the immunosuppressive effects of TGF-beta receptor signalling. Ann Rheum Dis. 2015;74:2224–35.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  35. 35.

    Osuka A, Hanschen M, Stoecklein V, Lederer JA. A protective role for inflammasome activation following injury. Shock. 2012;37:47–55.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. 36.

    Kim SH, Kim G, Han DH, Lee M, Kim I, Kim B, et al. Ezetimibe ameliorates steatohepatitis via AMP activated protein kinase-TFEB-mediated activation of autophagy and NLRP3 inflammasome inhibition. Autophagy. 2017;13:1767–81.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  37. 37.

    Wree A, McGeough MD, Pena CA, Schlattjan M, Li H, Inzaugarat ME, et al. NLRP3 inflammasome activation is required for fibrosis development in NAFLD. J Mol Med (Berl). 2014;92:1069–82.

    CAS  Article  Google Scholar 

  38. 38.

    Guarda G, Zenger M, Yazdi AS, Schroder K, Ferrero I, Menu P, et al. Differential expression of NLRP3 among hematopoietic cells. J Immunol. 2011;186:2529–34.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  39. 39.

    Bruchard M, Rebe C, Derangere V, Togbe D, Ryffel B, Boidot R, et al. The receptor NLRP3 is a transcriptional regulator of TH2 differentiation. Nat Immunol. 2015;16:859–70.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  40. 40.

    Camell CD, Sander J, Spadaro O, Lee A, Nguyen KY, Wing A, et al. Inflammasome-driven catecholamine catabolism in macrophages blunts lipolysis during ageing. Nature. 2017;550:119–23.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  41. 41.

    Jiang D, Chen S, Sun R, Zhang X, Wang D. The NLRP3 inflammasome: Role in metabolic disorders and regulation by metabolic pathways. Cancer Lett. 2018;419:8–19.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  42. 42.

    Liu XJ, Liu C, Zhu LY, Fan CL, Niu C, Liu XP, et al. Hepalatide ameliorated progression of nonalcoholic steatohepatitis in mice. Biomed Pharmacother. 2020;126:110053.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  43. 43.

    Prakash S, Rai U, Kosuru R, Tiwari V, Singh S. Amelioration of diet-induced metabolic syndrome and fatty liver with sitagliptin via regulation of adipose tissue inflammation and hepatic Adiponectin/AMPK levels in mice. Biochimie. 2020;168:198–209.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  44. 44.

    Vandanmagsar B, Youm YH, Ravussin A, Galgani JE, Stadler K, Mynatt RL, et al. The NLRP3 inflammasome instigates obesity-induced inflammation and insulin resistance. Nat Med. 2011;17:179–88.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  45. 45.

    Kotas ME, Jurczak MJ, Annicelli C, Gillum MP, Cline GW, Shulman GI, et al. Role of caspase-1 in regulation of triglyceride metabolism. Proc Natl Acad Sci USA. 2013;110:4810–5.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  46. 46.

    Smith U. Impaired (‘diabetic’) insulin signaling and action occur in fat cells long before glucose intolerance–is insulin resistance initiated in the adipose tissue? Int J Obes Relat Metab Disord. 2002;26:897–904.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  47. 47.

    Vergadi E, Ieronymaki E, Lyroni K, Vaporidi K, Tsatsanis C. Akt Signaling Pathway in Macrophage Activation and M1/M2 Polarization. J Immunol. 2017;198:1006–14.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  48. 48.

    Zhong S, Zhao L, Wang Y, Zhang C, Liu J, Wang P, et al. Cluster of differentiation 36 deficiency aggravates macrophage infiltration and hepatic inflammation by upregulating monocyte chemotactic protein-1 expression of hepatocytes through histone deacetylase 2-dependent pathway. Antioxid Redox Signal. 2017;27:201–14.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  49. 49.

    Schuster S, Cabrera D, Arrese M, Feldstein AE. Triggering and resolution of inflammation in NASH. Nat Rev Gastroenterol Hepatol. 2018;15:349–64.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  50. 50.

    Sun L, Ma W, Gao W, Xing Y, Chen L, Xia Z, et al. Propofol directly induces caspase-1-dependent macrophage pyroptosis through the NLRP3-ASC inflammasome. Cell Death Dis. 2019;10:542.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  51. 51.

    de Castro-Jorge LA, de Carvalho RVH, Klein TM, Hiroki CH, Lopes AH, Guimaraes RM, et al. The NLRP3 inflammasome is involved with the pathogenesis of Mayaro virus. PLoS Pathog. 2019;15:e1007934.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors are in debt to Associate Prof. Si Zhang in the Dept. of Biochemistry and Molecular Biology for providing the NLRP3 knock-out mice, and Prof. Xiuping Liu in the Dept. Pathology and Laboratory Medicine, School of Basic Medical Sciences, Fudan University for her generous assistance in pathologic evaluation of NAFLD activity score in H&E stained sections. The authors are grateful for technical assistance from the Technology Platform of Fudan University School of Basic Medical Sciences for the use of confocal microscope in this study.

Funding

This work is supported by the National Key R&D Program of China (#2016YFE0107400), the National Natural Science Foundation of China (NSFC #81272436, 81572356, 81871997), Shanghai Commission of Sciences and Technologies (#16140903700) to JW.

Author information

Affiliations

Authors

Contributions

L-YZ: Experimental design and conduct, data collection and analysis, manuscript preparation. CL, Z-RL: participating in experiment conduct and data analysis. CN: technical assistance and supervision. JW: Concept development, experimental design, data analysis, funding support, and manuscript preparation and finalization.

Corresponding author

Correspondence to Jian Wu.

Ethics declarations

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.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zhu, LY., Liu, C., Li, ZR. et al. NLRP3 deficiency did not attenuate NASH development under high fat calorie diet plus high fructose and glucose in drinking water. Lab Invest (2021). https://doi.org/10.1038/s41374-021-00535-3

Download citation

Search

Quick links