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Neutrophil extracellular traps promote acetaminophen-induced acute liver injury in mice via AIM2

Acta Pharmacologica Sinica (2024)Cite this article

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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Fig. 1: Excessive acetaminophen induces liver injury and apoptosis, pyroptosis and necroptosis in mice.
Fig. 2: Excessive acetaminophen induces hepatocyte PANoptosis via increased NETs formation in vitro.
Fig. 3: DNase1 treatment inhibited APAP-induced ALI as well as hepatocyte apoptosis, pyroptosis and necroptosis.
Fig. 4: Excessive acetaminophen induces the formation of PANoptosome and AIM2 pathway activation.
Fig. 5: Depletion of AIM2 results in reduced APAP-induced ALI and decreased formation of PANoptosome.
Fig. 6: mtDNA promoted the formation of NETs by TLR9-mediated signaling pathway.
Fig. 7: Excess APAP causes excess mtROS production and thus mtDNA leakage.

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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.

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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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Authors and Affiliations

  1. Inflammation and Immune Mediated Diseases Laboratory of Anhui Province, Anhui Institute of Innovative Drugs, School of Pharmacy, Anhui Medical University, Hefei, 230032, China

    Fan-le Zeng, Yuan Zhang, Zhong-hao Wang, Hui Zhang, Xue-teng Meng, Yi-qin Wu, Zhen-zhen Qian, Yu-hao Ding, Jun Li, Tao-tao Ma & Cheng Huang

  2. Anhui Provincial Institute of Translational Medicine, Hefei, 230032, China

    Tao-tao Ma & Cheng Huang

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Contributions

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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Correspondence to Tao-tao Ma or Cheng Huang.

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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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