Abstract
Activation of the inflammasome in hepatocytes and the liver-resident macrophages is associated with drug-induced hepatotoxicity and a plethora of metabolic diseases including nonalcoholic steatohepatitis (NASH). Initiation of this innate immune response requires two concomitant signals resulting in the formation of a molecular assembly that post-transcriptionally maturates a specific set of cytokines. While signal 1 results from the engagement and activation of pattern recognition receptors, signal 2 can be induced by diverse stimuli including adenosine triphosphate (ATP). Among various modules, NOD-like receptor 3 (NLRP3) inflammasome activation followed by caspase-1-dependent proIL-1β maturation has been observed in both preclinical models and NASH patients suggesting the crucial importance of inflammasome activation in NAFLD progression. The protocol reported here depicts an ex vivo method for investigating the role of inflammasome activation in macrophages and its impact on hepatocytes. We first described a rapid protocol for the isolation of primary Kupffer cells (KC) and hepatocytes from the murine liver. Next, to investigate the crosstalks between KCs and hepatocytes in the context of inflammasome activation, isolated KCs were activated with lipopolysaccharide (LPS), alone or in tandem with ATP, which resulted in inflammasome activation in KCs evident by abundant IL-1β secretion. Isolated primary hepatocytes were treated with conditioned medium (CM) from activated KCs to investigate the effect of inflammasome activation by various readouts. Moreover, this model also enabled us to investigate the role of specific cytokines by neutralizing them in the CM of inflammasome-activated KC. This precise ex vivo method provides a comprehensive protocol for investigating hepatocellular inflammasome activation.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Kesavardhana S, Kanneganti TD (2017) Mechanisms governing inflammasome activation, assembly and pyroptosis induction. Int Immunol 29:201–210
Martinon F, Burns K, Tschopp J (2002) The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-β. Mol Cell 10:417–426
Lopez-Castejon G, Brough D (2011) Understanding the mechanism of IL-1β secretion. Cytokine Growth Factor Rev 22:189–195
Dupont N, Jiang S, Pilli M, Ornatowski W, Bhattacharya D, Deretic V (2011) Autophagy-based unconventional secretory pathway for extracellular delivery of IL-1β. EMBO J 30:4701–4711
Lamkanfi M (2011) Emerging inflammasome effector mechanisms. Nat Rev Immunol 11:213–220
Fink SL, Cookson BT (2005) Apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells. Infect Immun 73:1907–1916
Berman N, Dunham J, Vivino F (2014) Cryopyrin-associated periodic fever syndrome presenting with fevers and serositis related to a novel NLRP3 gene mutation. J Clin Rheumatol 20:451–452
Place DE, Kanneganti TD (2018) Recent advances in inflammasome biology. Curr Opin Immunol 50:32–38
Hardy T, Oakley F, Anstee QM, Day CP (2016) Nonalcoholic fatty liver disease: pathogenesis and disease spectrum. Annu Rev Pathol Mech Dis 11:451–496
Feldstein AE, Canbay A, Angulo P, Taniai M, Burgart LJ, Lindor KD, Gores GJ (2003) Hepatocyte apoptosis and Fas expression are prominent features of human nonalcoholic steatohepatitis. Gastroenterology 125:437–443
Kubes P, Mehal WZ (2012) Sterile inflammation in the liver. Gastroenterology 143:1158–1172
Zeng T, Zhang CL, Xiao M, Yang R, Xie KQ (2016) Critical roles of kupffer cells in the pathogenesis of alcoholic liver disease: from basic science to clinical trials. Front Immunol 7:1
Enomoto N, Yamashina S, Kono H, Schemmer P, Rivera CA, Enomoto A, Nishiura T, Nishimura T, Brenner DA, Thurman RG (1999) Development of a new, simple rat model of early alcohol-induced liver injury based on sensitization of kupffer cells. Hepatology 29:1680–1689
Szabo G, Petrasek J (2015) Inflammasome activation and function in liver disease. Nat Rev Gastroenterol Hepatol 12:387–400
Weischenfeldt J, Porse B (2008) Bone marrow-derived macrophages (BMM): isolation and applications. CSH Protoc 2008:pdb.prot5080
Bailey JD, Shaw A, McNeill E, Nicol T, Diotallevi M, Chuaiphichai S, Patel J, Hale A, Channon KM, Crabtree MJ (2020) Isolation and culture of murine bone marrow-derived macrophages for nitric oxide and redox biology. Nitric Oxide 100–101:17–29
Adak M, Das D, Niyogi S, Nagalakshmi C, Ray D, Chakrabarti P (2018) Inflammasome activation in Kupffer cells confers a prostective response in nonalcoholic steatohepatitis through pigment epithelium-derived factor expression. FASEB J 32:6510–6524
Acknowledgments
We sincerely thank Avishek Paul for the help in writing the protocols, and Sujay Krishna Maity for drawing cliparts for the figures. This work has been supported by grants to P.C. by the Indian Council of Medical Research (ICMR), India (5/4/5-9/Diab.-16-NCD-II) and Council of Scientific and Industrial Research (CSIR), India (MLP125 and MLP138). M.A. and D.D. received a research fellowship from University Grants Commission (UGC), India.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Das, D., Adak, M., Chakrabarti, P. (2022). Ex Vivo Dual-Hit Method for Inflammasome Activation in Liver. In: Sarkar, D. (eds) Non-Alcoholic Steatohepatitis. Methods in Molecular Biology, vol 2455. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2128-8_20
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2128-8_20
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2127-1
Online ISBN: 978-1-0716-2128-8
eBook Packages: Springer Protocols