Skip to main content
Log in

NLRP3 inflammasome activation is required for fibrosis development in NAFLD

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

Abstract

NLR inflammasomes, caspase 1 activation platforms critical for processing key pro-inflammatory cytokines, have been implicated in the development of nonalcoholic fatty liver disease (NAFLD). As the direct role of the NLRP3 inflammasome remains unclear, we tested effects of persistent NLRP3 activation as a contributor to NAFLD development and, in particular, as a modulator of progression from benign hepatic steatosis to steatohepatitis during diet-induced NAFLD. Gain of function tamoxifen-inducible Nlrp3 knock-in mice allowing for in vivo temporal control of NLRP3 activation and loss of function Nlrp3 knockout mice were placed on short-term choline-deficient amino acid-defined (CDAA) diet, to induce isolated hepatic steatosis or long-term CDAA exposure, to induce severe steatohepatitis and fibrosis, respectively. Expression of NLRP3 associated proteins was assessed in liver biopsies of a well-characterized group of patients with the full spectrum of NAFLD. Nlrp3 −/− mice were protected from long-term feeding CDAA-induced hepatomegaly, liver injury, and infiltration of activated macrophages. More importantly, Nlrp3 −/− mice showed marked protection from CDAA-induced liver fibrosis. After 4 weeks on CDAA diet, wild-type (WT) animals showed isolated hepatic steatosis while Nlrp3 knock-in mice showed severe liver inflammation, with increased infiltration of activated macrophages and early signs of liver fibrosis. In the liver samples of patients with NAFLD, inflammasome components were significantly increased in those patients with nonalcoholic steatohepatitis (NASH) when compared to those with non-NASH NAFLD with mRNA levels of pro-IL1 beta correlated to levels of COL1A1. Our study uncovers a crucial role for the NLRP3 inflammasome in the development of NAFLD. These findings may lead to novel therapeutic strategies aimed at halting the progression of hepatic steatosis to the more severe forms of this disease.

Key message

  • Mice with NLRP3 inflammasome loss of function are protected from diet-induced steatohepatitis.

  • NLRP3 inflammasome gain of function leads to early and severe onset of diet-induced steatohepatitis in mice.

  • Patients with severe NAFLD exhibit increased levels of NLRP3 inflammasome components and levels of pro-IL1β mRNA correlate with the expression of COL1A1.

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

Access this article

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
Fig. 6

Similar content being viewed by others

Abbreviations

α-SMA:

Alpha smooth muscle actin

ALT:

Alanine aminotransferase

ASC:

Apoptosis-associated speck-like protein containing a caspase recruitment domain

Arg1:

Arginase 1

ASH:

Alcoholic steatohepatitis

BMI:

Body mass index

Casp1:

Caspase 1

CDAA:

Choline-deficient amino acid-defined

COL1A1:

Collagen, type I, alpha 1

CSAA:

Choline-supplemented amino acid-defined

CTGF:

Connective tissue growth factor

CXCL 2:

Chemokine (C-X-C motif) ligand 2

DAMPs:

Damage associated molecular patterns

F4/80:

Murine macrophage marker

HSC:

Hepatic stellate cell

ICAM1:

Intercellular adhesion molecule 1

IL:

Interleukin

iNOS:

Inducible form of nitric oxide synthase

Ly6c:

Lymphocyte antigen 6 complex

MCP1:

Monocyte chemotactic protein-1

MMP2:

Matrix metalloproteinase-2

MPO:

Myeloperoxidase

NAFLD:

Nonalcoholic fatty liver disease

NASH:

Nonalcoholic steatohepatitis

NC:

Normal chow

NLRs:

Nucleotide-binding oligomerization domain (NOD) leucine-rich-repeat containing receptors

PAMPs:

Pathogen associated molecular patterns

PBS:

Phosphate-buffered saline

TIMP1:

Tissue inhibitor of matrix metalloproteinase 1

TNF-α:

Tumor necrosis factor alpha

WT:

Wild type

References

  1. Levene AP, Goldin RD (2012) The epidemiology, pathogenesis and histopathology of fatty liver disease. Histopathology 61:141–152

    Article  PubMed  Google Scholar 

  2. Vernon G, Baranova A, Younossi ZM (2011) Systematic review: the epidemiology and natural history of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis in adults. Aliment Pharmacol Ther 34:274–285

    Article  PubMed  CAS  Google Scholar 

  3. Mahady SE, George J (2012) Management of nonalcoholic steatohepatitis: an evidence-based approach. Clin Liver Dis 16:631–645

    Article  PubMed  Google Scholar 

  4. Adams LA, Lymp JF, St Sauver J, Sanderson SO, Lindor KD, Feldstein A, Angulo P (2005) The natural history of nonalcoholic fatty liver disease: a population-based cohort study. Gastroenterology 129:113–121

    Article  PubMed  Google Scholar 

  5. Matteoni CA, Younossi ZM, Gramlich T, Boparai N, Liu YC, McCullough AJ (1999) Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity. Gastroenterology 116:1413–1419

    Article  PubMed  CAS  Google Scholar 

  6. Brunt EM (2002) Alcoholic and nonalcoholic steatohepatitis. Clin Liver Dis 6:399–420, vii

    Article  PubMed  Google Scholar 

  7. Hjelkrem MC, Torres DM, Harrison SA (2008) Nonalcoholic fatty liver disease. Minerva Med 99:583–593

    PubMed  CAS  Google Scholar 

  8. Bechmann LP, Hannivoort RA, Gerken G, Hotamisligil GS, Trauner M, Canbay A (2012) The interaction of hepatic lipid and glucose metabolism in liver diseases. J Hepatol 56:952–964

    Article  PubMed  CAS  Google Scholar 

  9. Wree A, Kahraman A, Gerken G, Canbay A (2011) Obesity affects the liver—the link between adipocytes and hepatocytes. Digestion 83:124–133

    Article  PubMed  Google Scholar 

  10. Tilg H, Moschen AR (2010) Evolution of inflammation in nonalcoholic fatty liver disease: the multiple parallel hits hypothesis. Hepatology 52:1836–1846

    Article  PubMed  CAS  Google Scholar 

  11. Martinon F, Burns K, Tschopp J (2002) The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Mol Cell 10:417–426

    Article  PubMed  CAS  Google Scholar 

  12. Netea MG, van der Meer JW (2011) Immunodeficiency and genetic defects of pattern-recognition receptors. N Engl J Med 364:60–70

    Article  PubMed  CAS  Google Scholar 

  13. Takeuchi O, Akira S (2010) Pattern recognition receptors and inflammation. Cell 140:805–820

    Article  PubMed  CAS  Google Scholar 

  14. Szabo G, Csak T (2012) Inflammasomes in liver diseases. J Hepatol 57:642–654

    Article  PubMed  CAS  Google Scholar 

  15. Kubes P, Mehal WZ (2012) Sterile inflammation in the liver. Gastroenterology 143:1158–1172

    Article  PubMed  CAS  Google Scholar 

  16. Kamari Y, Shaish A, Vax E, Shemesh S, Kandel-Kfir M, Arbel Y, Olteanu S, Barshack I, Dotan S, Voronov E et al (2011) Lack of interleukin-1alpha or interleukin-1beta inhibits transformation of steatosis to steatohepatitis and liver fibrosis in hypercholesterolemic mice. J Hepatol 55:1086–1094

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  17. de Roos B, Rungapamestry V, Ross K, Rucklidge G, Reid M, Duncan G, Horgan G, Toomey S, Browne J, Loscher CE et al (2009) Attenuation of inflammation and cellular stress-related pathways maintains insulin sensitivity in obese type I interleukin-1 receptor knockout mice on a high-fat diet. Proteomics 9:3244–3256

    Article  PubMed  Google Scholar 

  18. Dixon LJ, Berk M, Thapaliya S, Papouchado BG, Feldstein AE (2012) Caspase-1-mediated regulation of fibrogenesis in diet-induced steatohepatitis. Lab Investig J Tech Methods Pathol 92:713–723

    Article  CAS  Google Scholar 

  19. Wree A, Eguchi A, McGeough MD, Pena CA, Johnson CD, Canbay A, Hoffman HM, Feldstein AE (2013) NLRP3 inflammasome activation results in hepatocyte pyroptosis, liver inflammation and fibrosis. Hepatology 59:898–910

  20. Kodama Y, Kisseleva T, Iwaisako K, Miura K, Taura K, De Minicis S, Osterreicher CH, Schnabl B, Seki E, Brenner DA (2009) c-Jun N-terminal kinase-1 from hematopoietic cells mediates progression from hepatic steatosis to steatohepatitis and fibrosis in mice. Gastroenterology 137:1467–1477, e1465

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  21. Sutterwala FS, Ogura Y, Szczepanik M, Lara-Tejero M, Lichtenberger GS, Grant EP, Bertin J, Coyle AJ, Galan JE, Askenase PW et al (2006) Critical role for NALP3/CIAS1/Cryopyrin in innate and adaptive immunity through its regulation of caspase-1. Immunity 24:317–327

    Article  PubMed  CAS  Google Scholar 

  22. Brydges SD, Mueller JL, McGeough MD, Pena CA, Misaghi A, Gandhi C, Putnam CD, Boyle DL, Firestein GS, Horner AA et al (2009) Inflammasome-mediated disease animal models reveal roles for innate but not adaptive immunity. Immunity 30:875–887

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  23. Hayashi S, McMahon AP (2002) Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse. Dev Biol 244:305–318

    Article  PubMed  CAS  Google Scholar 

  24. McGeough MD, Pena CA, Mueller JL, Pociask DA, Broderick L, Hoffman HM, Brydges SD (2012) Cutting edge: IL-6 is a marker of inflammation with no direct role in inflammasome-mediated mouse models. J Immunol 189:2707–2711

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  25. Nakae D, Yoshiji H, Mizumoto Y, Horiguchi K, Shiraiwa K, Tamura K, Denda A, Konishi Y (1992) High incidence of hepatocellular carcinomas induced by a choline deficient L-amino acid defined diet in rats. Cancer Res 52:5042–5045

    PubMed  CAS  Google Scholar 

  26. Kahraman A, Sowa JP, Schlattjan M, Sydor S, Pronadl M, Wree A, Beilfuss A, Kilicarslan A, Altinbas A, Bechmann LP et al (2013) Fetuin-A mRNA expression is elevated in NASH compared with NAFL patients. Clin Sci 125:391–400

    Article  PubMed  CAS  Google Scholar 

  27. Kalsch J, Bechmann LP, Kalsch H, Schlattjan M, Erhard J, Gerken G, Canbay A (2011) Evaluation of biomarkers of NAFLD in a cohort of morbidly obese patients. J Nutr Metab 2011: 369168. doi:10.1155/2011/369168

  28. Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, Ferrell LD, Liu YC, Torbenson MS, Unalp-Arida A et al (2005) Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 41:1313–1321

    Article  PubMed  Google Scholar 

  29. Petrasek J, Bala S, Csak T, Lippai D, Kodys K, Menashy V, Barrieau M, Min SY, Kurt-Jones EA, Szabo G (2012) IL-1 receptor antagonist ameliorates inflammasome-dependent alcoholic steatohepatitis in mice. J Clin Invest 122:3476–3489

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  30. Miura K, Kodama Y, Inokuchi S, Schnabl B, Aoyama T, Ohnishi H, Olefsky JM, Brenner DA, Seki E (2010) Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1beta in mice. Gastroenterology 139(323–334):e327

    Google Scholar 

  31. Dixon LJ, Flask CA, Papouchado BG, Feldstein AE, Nagy LE (2013) Caspase-1 as a central regulator of high fat diet-induced non-alcoholic steatohepatitis. PLoS One 8:e56100

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  32. Watanabe A, Sohail MA, Gomes DA, Hashmi A, Nagata J, Sutterwala FS, Mahmood S, Jhandier MN, Shi Y, Flavell RA et al (2009) Inflammasome-mediated regulation of hepatic stellate cells. Am J Physiol Gastrointest Liver Physiol 296:G1248–G1257

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  33. Wen H, Gris D, Lei Y, Jha S, Zhang L, Huang MT, Brickey WJ, Ting JP (2011) Fatty acid-induced NLRP3-ASC inflammasome activation interferes with insulin signaling. Nat Immunol 12:408–415

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  34. Vandanmagsar B, Youm YH, Ravussin A, Galgani JE, Stadler K, Mynatt RL, Ravussin E, Stephens JM, Dixit VD (2011) The NLRP3 inflammasome instigates obesity-induced inflammation and insulin resistance. Nat Med 17:179–188

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  35. Henao-Mejia J, Elinav E, Jin C, Hao L, Mehal WZ, Strowig T, Thaiss CA, Kau AL, Eisenbarth SC, Jurczak MJ et al (2012) Inflammasome-mediated dysbiosis regulates progression of NAFLD and obesity. Nature 482:179–185

    PubMed  CAS  PubMed Central  Google Scholar 

  36. Eisener-Dorman AF, Lawrence DA, Bolivar VJ (2009) Cautionary insights on knockout mouse studies: the gene or not the gene? Brain Behav Immun 23:318–324

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  37. Davey RA, MacLean HE (2006) Current and future approaches using genetically modified mice in endocrine research. Am J Physiol Endocrinol Metab 291:E429–E438

    Article  PubMed  CAS  Google Scholar 

  38. Csak T, Ganz M, Pespisa J, Kodys K, Dolganiuc A, Szabo G (2011) Fatty acid and endotoxin activate inflammasomes in mouse hepatocytes that release danger signals to stimulate immune cells. Hepatology 54:133–144

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  39. Rizki G, Arnaboldi L, Gabrielli B, Yan J, Lee GS, Ng RK, Turner SM, Badger TM, Pitas RE, Maher JJ (2006) Mice fed a lipogenic methionine-choline-deficient diet develop hypermetabolism coincident with hepatic suppression of SCD-1. J Lipid Res 47:2280–2290

    Article  PubMed  CAS  Google Scholar 

  40. Oosterveer MH, van Dijk TH, Tietge UJ, Boer T, Havinga R, Stellaard F, Groen AK, Kuipers F, Reijngoud DJ (2009) High fat feeding induces hepatic fatty acid elongation in mice. PLoS One 4:e6066

    Article  PubMed  PubMed Central  Google Scholar 

  41. de Almeida IT, Cortez-Pinto H, Fidalgo G, Rodrigues D, Camilo ME (2002) Plasma total and free fatty acids composition in human non-alcoholic steatohepatitis. Clin Nutr 21:219–223

    Article  PubMed  Google Scholar 

  42. Puri P, Wiest MM, Cheung O, Mirshahi F, Sargeant C, Min HK, Contos MJ, Sterling RK, Fuchs M, Zhou H et al (2009) The plasma lipidomic signature of nonalcoholic steatohepatitis. Hepatology 50:1827–1838

    Article  PubMed  CAS  Google Scholar 

  43. Wree A, Bechmann LP, Claudel T, Schlattjan M, Sowa JP, Baba H, Gerken G, Feldstein AE, Trauner M, Canbay A (2013) Bariatric surgery reduces adipocyte size, improves liver injury and counteracts lipotoxicity via changes in serum fatty acid composition and adiponectin levels. J Hepatol 58:S553

Download references

Acknowledgments

We thank Martin Pronadl and Rudolf Ott from the Clinic of Surgery II at Alfried Krupp Hospital Essen, Germany, for collecting tissue and serum samples during bariatric surgeries and clinical follow up of the enrolled patients. We thank Bettina Papouchado for assessing steatosis, inflammation, and ballooning in the liver samples. This work was funded by NIH (DK076852 and DK082451 to AEF and AI52430 to HMH) and the German Research Foundation (DFG-grant 173/2-1 to AW).

Conflict of interest

The authors state that they have nothing to disclose.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Ariel E. Feldstein.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplemental Table 1

(DOCX 17 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wree, A., McGeough, M.D., Peña, C.A. et al. NLRP3 inflammasome activation is required for fibrosis development in NAFLD. J Mol Med 92, 1069–1082 (2014). https://doi.org/10.1007/s00109-014-1170-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00109-014-1170-1

Keywords

Navigation