Abstract
Excessive acetaminophen (APAP) can induce neutrophil activation and hepatocyte death. Along with hepatocyte dysfunction and death, NETosis (a form of neutrophil-associated inflammation) plays a vital role in the progression of acute liver injury (ALI) induced by APAP overdose. It has been shown that activated neutrophils tend to migrate towards the site of injury and participate in inflammatory processes via formation of neutrophil extracellular traps (NETs). In this study we investigated whether NETs were involved in hepatocyte injury and contributed to APAP-induced ALI progression. ALI mouse model was established by injecting overdose (350 mg/kg) of APAP. After 24 h, blood and livers were harvested for analyses. We showed that excessive APAP induced multiple programmed cell deaths of hepatocytes including pyroptosis, apoptosis and necroptosis, accompanied by significantly increased NETs markers (MPO, citH3) in the liver tissue and serum. Preinjection of DNase1 (10 U, i.p.) for two consecutive days significantly inhibited NETs formation, reduced PANoptosis and consequently alleviated excessive APAP-induced ALI. In order to clarify the communication between hepatocytes and neutrophils, we induced NETs formation in isolated neutrophils, and treated HepaRG cells with NETs. We found that NETs treatment markedly increased the activation of GSDMD, caspase-3 and MLKL, while pre-treatment with DNase1 down-regulated the expression of these proteins. Knockdown of AIM2 (a cytosolic innate immune receptor) abolished NETs-induced PANoptosis in HepaRG cells. Furthermore, excessive APAP-associated ALI was significantly attenuated in AIM2KO mice, and PANoptosis occurred less frequently. Upon restoring AIM2 expression in AIM2KO mice using AAV9 virus, both hepatic injury and PANoptosis was aggravated. In addition, we demonstrated that excessive APAP stimulated mtROS production and mitochondrial DNA (mtDNA) leakage, and mtDNA activated the TLR9 pathway to promote NETs formation. Our results uncover a novel mechanism of NETs and PANoptosis in APAP-associated ALI, which might serve as a therapeutic target.
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Data availability
All data generated or analyzed during this study are included in the manuscript. Further inquiries should be directed to the corresponding author.
Materials availability
Mice generated in this study will be made available upon request. We may require a payment and/or a completed Materials Transfer Agreement in case there is potential for commercial application.
References
Jaeschke H. Acetaminophen: dose-dependent drug hepatotoxicity and acute liver failure in patients. Dig Dis. 2015;33:464–71.
McGill MR, Hinson JA. The development and hepatotoxicity of acetaminophen: reviewing over a century of progress. Drug Metab Rev. 2020;52:472–500.
Ortland I, Mirjalili M, Kullak-Ublick GA, Peymani P. Drug-induced liver injury in Switzerland: an analysis of drug-related hepatic disorders in the WHO pharmacovigilance database VigiBase from 2010 to 2020. Swiss Med Wkly. 2021;151:w20503.
Santos G, Figueira ERR, D’Albuquerque LAC, Lisboa PB, de Almeida MD, Filgueira NA, et al. Evaluation of drug-induced liver injury as etiology for acute liver failure in Brazil. Ann Hepatol. 2021;23:100310.
Darr U, Sussman NL. Drug-induced liver injury in the setting of analgesic use. Clin Liver Dis. 2020;24:121–9.
Chayanupatkul M, Schiano TD. Acute liver failure secondary to drug-induced liver injury. Clin Liver Dis. 2020;24:75–87.
Liu J, Jiang M, Jin Q, Wu YL, Cui ZY, Cui BW, et al. Modulation of HMGB1 release in APAP-induced liver injury: a possible strategy of Chikusetsusaponin V targeting NETs formation. Front Pharmacol. 2021;12:723881.
Marques PE, Oliveira AG, Pereira RV, David BA, Gomides LF, Saraiva AM, et al. Hepatic DNA deposition drives drug-induced liver injury and inflammation in mice. Hepatology. 2015;61:348–60.
Papackova Z, Heczkova M, Dankova H, Sticova E, Lodererova A, Bartonova L, et al. Silymarin prevents acetaminophen-induced hepatotoxicity in mice. PLoS One. 2018;13:e0191353.
Chen L, Dong J, Liao S, Wang S, Wu Z, Zuo M, et al. Loss of Sam50 in hepatocytes induces cardiolipin-dependent mitochondrial membrane remodeling to trigger mtDNA release and liver injury. Hepatology. 2022;76:1389–408.
Ghanem CI, Perez MJ, Manautou JE, Mottino AD. Acetaminophen from liver to brain: New insights into drug pharmacological action and toxicity. Pharmacol Res. 2016;109:119–31.
He Y, Feng D, Li M, Gao Y, Ramirez T, Cao H, et al. Hepatic mitochondrial DNA/Toll-like receptor 9/MicroRNA-223 forms a negative feedback loop to limit neutrophil overactivation and acetaminophen hepatotoxicity in mice. Hepatology. 2017;66:220–34.
Zhao J, Kim JW, Zhou Z, Qi J, Tian W, Lim CW, et al. Macrophage-Inducible C-type lectin signaling exacerbates acetaminophen-induced liver injury by promoting Kupffer cell activation in mice. Mol Pharmacol. 2021;99:92–103.
Liu ZX, Govindarajan S, Kaplowitz N. Innate immune system plays a critical role in determining the progression and severity of acetaminophen hepatotoxicity. Gastroenterology. 2004;127:1760–74.
Guo H, Chen S, Xie M, Zhou C, Zheng M. The complex roles of neutrophils in APAP-induced liver injury. Cell Prolif. 2021;54:e13040.
Goldberger A, Hnilica LS, Casey SB, Briggs RC. Properties of a nuclear protein marker of human myeloid cell differentiation. J Biol Chem. 1986;261:4726–31.
Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, et al. Neutrophil extracellular traps kill bacteria. Science. 2004;303:1532–5.
Huang H, Tohme S, Al-Khafaji AB, Tai S, Loughran P, Chen L, et al. Damage-associated molecular pattern-activated neutrophil extracellular trap exacerbates sterile inflammatory liver injury. Hepatology. 2015;62:600–14.
Zhao M, Wang Y, Li L, Liu S, Wang C, Yuan Y, et al. Mitochondrial ROS promote mitochondrial dysfunction and inflammation in ischemic acute kidney injury by disrupting TFAM-mediated mtDNA maintenance. Theranostics. 2021;11:1845–63.
Zheng F, Ma L, Li X, Wang Z, Gao R, Peng C, et al. Neutrophil extracellular traps induce glomerular endothelial cell dysfunction and pyroptosis in diabetic kidney disease. Diabetes. 2022;71:2739–50.
Zhang H, Liu J, Zhou Y, Qu M, Wang Y, Guo K, et al. Neutrophil extracellular traps mediate m6A modification and regulates sepsis-associated acute lung injury by activating ferroptosis in alveolar epithelial cells. Int J Biol Sci. 2022;18:3337–57.
Zhang Y, Xue W, Zhang W, Yuan Y, Zhu X, Wang Q, et al. Histone methyltransferase G9a protects against acute liver injury through GSTP1. Cell Death Differ. 2019;27:1243–58.
Eaton JS, Lin ZP, Sartorelli AC, Bonawitz ND, Shadel GS. Ataxia-telangiectasia mutated kinase regulates ribonucleotide reductase and mitochondrial homeostasis. J Clin Invest. 2007;117:2723–34.
Malik AN, Shahni R, Rodriguez-de-Ledesma A, Laftah A, Cunningham P. Mitochondrial DNA as a non-invasive biomarker: Accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias. Biochem Biophys Res Commun. 2011;412:1–7.
Man SM, Karki R, Kanneganti TD. AIM2 inflammasome in infection, cancer, and autoimmunity: Role in DNA sensing, inflammation, and innate immunity. Eur J Immunol. 2016;46:269–80.
Zhao M, Zhou Y, Liu S, Li L, Chen Y, Cheng J, et al. Control release of mitochondria-targeted antioxidant by injectable self-assembling peptide hydrogel ameliorated persistent mitochondrial dysfunction and inflammation after acute kidney injury. Drug Deliv. 2018;25:546–54.
Zhou Y, Fan X, Jiao T, Li W, Chen P, Jiang Y, et al. SIRT6 as a key event linking P53 and NRF2 counteracts APAP-induced hepatotoxicity through inhibiting oxidative stress and promoting hepatocyte proliferation. Acta Pharm Sin B. 2021;11:89–99.
Block H, Rossaint J, Zarbock A. The fatal circle of NETs and NET-associated DAMPs contributing to organ dysfunction. Cells-Basel. 2022;11:1919.
Lee S, Karki R, Wang Y, Nguyen LN, Kalathur RC, Kanneganti TD. AIM2 forms a complex with pyrin and ZBP1 to drive PANoptosis and host defence. Nature. 2021;597:415–9.
Liu Z, Wang M, Wang X, Bu Q, Wang Q, Su W, et al. XBP1 deficiency promotes hepatocyte pyroptosis by impairing mitophagy to activate mtDNA-cGAS-STING signaling in macrophages during acute liver injury. Redox Biol. 2022;52:102305.
Niu B, Lei X, Xu Q, Ju Y, Xu D, Mao L, et al. Protecting mitochondria via inhibiting VDAC1 oligomerization alleviates ferroptosis in acetaminophen-induced acute liver injury. Cell Biol Toxicol. 2022;38:505–30.
Ye H, Chen C, Wu H, Zheng K, Martin-Adrados B, Caparros E, et al. Genetic and pharmacological inhibition of XBP1 protects against APAP hepatotoxicity through the activation of autophagy. Cell Death Dis. 2022;13:143.
Hussain S, Ashafaq M, Alshahrani S, Siddiqui R, Ahmed RA, Khuwaja G, et al. Cinnamon oil against acetaminophen-induced acute liver toxicity by attenuating inflammation, oxidative stress and apoptosis. Toxicol Rep. 2020;7:1296–304.
Ramachandran A, Jaeschke H. Acetaminophen hepatotoxicity: a mitochondrial perspective. Adv Pharmacol. 2019;85:195–219.
Wang J, Zhang L, Shi Q, Yang B, He Q, Wang J, et al. Targeting innate immune responses to attenuate acetaminophen-induced hepatotoxicity. Biochem Pharmacol. 2022;202:115142.
Zhang C, Shi X, Su Z, Hu C, Mu X, Pan J, et al. CD36 deficiency ameliorates drug-induced acute liver injury in mice. Mol Med. 2021;27:57.
Marques PE, Amaral SS, Pires DA, Nogueira LL, Soriani FM, Lima BH, et al. Chemokines and mitochondrial products activate neutrophils to amplify organ injury during mouse acute liver failure. Hepatology. 2012;56:1971–82.
Pinegin B, Vorobjeva N, Pinegin V. Neutrophil extracellular traps and their role in the development of chronic inflammation and autoimmunity. Autoimmun Rev. 2015;14:633–40.
Aube FA, Bidias A, Pepin G. Who and how, DNA sensors in NETs-driven inflammation. Front Immunol. 2023;14:1190177.
Zhao J, Zhen N, Zhou Q, Lou J, Cui W, Zhang G, et al. NETs promote inflammatory injury by activating cGAS-STING pathway in acute lung injury. Int J Mol Sci. 2023;24:5125.
Shojaie L, Iorga A, Dara L. Cell death in liver diseases: a review. Int J Mol Sci. 2020;21:9682.
Zheng M, Kanneganti TD. The regulation of the ZBP1‐NLRP3 inflammasome and its implications in pyroptosis, apoptosis, and necroptosis (PANoptosis). Immunol Rev. 2020;297:26–38.
Fernandes-Alnemri T, Yu JW, Datta P, Wu J, Alnemri ES. AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA. Nature. 2009;458:509–13.
Hornung V, Ablasser A, Charrel-Dennis M, Bauernfeind F, Horvath G, Caffrey DR, et al. AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC. Nature. 2009;458:514–8.
Castanheira FVS, Kubes P. Neutrophils and NETs in modulating acute and chronic inflammation. Blood. 2019;133:2178–85.
Honda M, Kubes P. Neutrophils and neutrophil extracellular traps in the liver and gastrointestinal system. Nat Rev Gastroenterol Hepatol. 2018;15:206–21.
Wang H, Li T, Chen S, Gu Y, Ye S. Neutrophil extracellular trap mitochondrial DNA and its autoantibody in systemic lupus erythematosus and a proof-of-concept trial of metformin. Arthritis Rheumatol. 2015;67:3190–200.
Zhang X, Wu J, Liu Q, Li X, Li S, Chen J, et al. mtDNA-STING pathway promotes necroptosis-dependent enterocyte injury in intestinal ischemia reperfusion. Cell Death Dis. 2020;11:1050.
Zhao X, Yu M, Zhao Y, Zheng Y, Meng L, Du K, et al. Circulating cell-free mtDNA release is associated with the activation of cGAS-STING pathway and inflammation in mitochondrial diseases. J Neurol. 2022;269:4985–96.
Itagaki K, Kaczmarek E, Lee YT, Tang IT, Isal B, Adibnia Y, et al. Mitochondrial DNA released by trauma induces neutrophil extracellular traps. PLoS One. 2015;10:e0120549.
Sun S, Duan Z, Wang X, Chu C, Yang C, Chen F, et al. Neutrophil extracellular traps impair intestinal barrier functions in sepsis by regulating TLR9-mediated endoplasmic reticulum stress pathway. Cell Death Dis. 2021;12:606.
Acknowledgements
This work was supported by funding from National Natural Science Foundation of China (U19A2001), Anhui Fund for Distinguished Young Scholars (2022AH020050), the Scientific Research Promoti on Fund of Anhui Medical University (2022xkjT010), Scientific Reasearch Platform Improvement Project of Anhui Medical University (2023xkjT049) and Research Fund of Anhui Institute of translational medicine (2021zhyx-B06, 2022zhyx-B07). The authors thank the Center for Scientific Research of Anhui Medical University for valuable help in our experiment.
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FLZ performed most of the experiments, analyzed the data and wrote the paper. CH and TTM are mainly responsible for the design, supervision, and editing of the manuscript for this experiment. YZ, YHD and ZHW provided a series of experimental instructions and proposals. HZ, XTM, YQW and YZ helped with the animal experiments. ZZQ helped with cell experiments. JL designed the experiments. All authors approved the final version of the manuscript.
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Zeng, Fl., Zhang, Y., Wang, Zh. et al. Neutrophil extracellular traps promote acetaminophen-induced acute liver injury in mice via AIM2. Acta Pharmacol Sin (2024). https://doi.org/10.1038/s41401-024-01239-2
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DOI: https://doi.org/10.1038/s41401-024-01239-2