Elsevier

Journal of Hepatology

Volume 68, Issue 4, April 2018, Pages 773-782
Journal of Hepatology

Research Article
Gasdermin D plays a key role as a pyroptosis executor of non-alcoholic steatohepatitis in humans and mice

https://doi.org/10.1016/j.jhep.2017.11.040Get rights and content

Highlights

  • Hepatic N-terminal cleavage fragments of GSDMD (GSDMD-N) are associated with lobular inflammation and hepatic ballooning.

  • GSDMD-N is a potential biomarker for the diagnosis of non-alcoholic steatohepatitis.

  • GSDMD plays a key role in steatohepatitis by mediating macrophage infiltration, NF-ĸB activation and lipogenesis.

Background & Aims

Gasdermin D (GSDMD)-executed programmed necrosis is involved in inflammation and controls interleukin (IL)-1β release. However, the role of GSDMD in non-alcoholic steatohepatitis (NASH) remains unclear. We investigated the role of GSDMD in the pathogenesis of steatohepatitis.

Methods

Human liver tissues from patients with non-alcoholic fatty liver disease (NAFLD) and control individuals were obtained to evaluate GSDMD expression. Gsdmd knockout (Gsdmd−/−) mice, obese db/db mice and their wild-type (WT) littermates were fed with methionine-choline deficient (MCD) or control diet to induce steatohepatitis. The Gsdmd−/− and WT mice were also used in a high-fat diet (HFD)-induced NAFLD model. In addition, Alb-Cre mice were administered an adeno-associated virus (AAV) vector that expressed the gasdermin-N domain (AAV9-FLEX-GSDMD-N) and were fed with either MCD or control diet for 10 days.

Results

GSDMD and its pyroptosis-inducing fragment GSDMD-N were upregulated in liver tissues of human NAFLD/NASH. Importantly, hepatic GSDMD-N protein levels were significantly higher in human NASH and correlated with the NAFLD activity score and fibrosis. GSDMD-N remained a potential biomarker for the diagnosis of NASH. MCD-fed Gsdmd−/− mice exhibit decreased severity of steatosis and inflammation compared with WT littermates. GSDMD was associated with the secretion of pro-inflammatory cytokines (IL-1β, TNF-α, and MCP-1 [CCL2]) and persistent activation of the NF-ĸB signaling pathway. Gsdmd−/− mice showed lower steatosis, mainly because of reduced expression of the lipogenic gene Srebp1c (Srebf1) and upregulated expression of lipolytic genes, including Pparα, Aco [Klk15], Lcad [Acadl], Cyp4a10 and Cyp4a14. Alb-Cre mice administered with AAV9-FLEX-GSDMD-N showed significantly aggravated steatohepatitis when fed with MCD diet.

Conclusion

As an executor of pyroptosis, GSDMD plays a key role in the pathogenesis of steatohepatitis, by controlling cytokine secretion, NF-ĸB activation, and lipogenesis.

Lay summary

Non-alcoholic fatty liver disease has become one of the most feared chronic liver diseases, because it is the most rapidly growing indication for adult liver transplantation and a major cause of hepatocellular carcinoma. However, the mechanisms involved in the transformation of simple steatosis to steatohepatitis remain unclear. Herein, we show that gasdermin D driven pyroptosis is prominent in patients with non-alcoholic steatohepatitis (NASH), and gasdermin-N domain remains a potential biomarker for the diagnosis of NASH. Gasdermin D plays a key role in the pathogenesis of NASH by regulating lipogenesis, the inflammatory response, and the NF-ĸB signaling pathway, revealing potential treatment targets for NASH in humans.

Introduction

Non-alcoholic fatty liver disease (NAFLD) represents a multi-step biological disorder in the liver, increasing the risk of cirrhosis and tumorigenesis.[1], [2] The key aspects of steatohepatitis have been precisely mimicked by extensive basic and translation research. This has enabled the reductionist assessment of genes and dietary factors involved in the pathogenesis of NAFLD.[3], [4] Toxic lipid accumulation in the liver acts as the primary insult which initiates and propagates damage, leading to hepatocyte injury and resultant inflammation.[5], [6] It is important to note that inflammation in the liver is believed to be the compelling feature that transforms simple steatosis to steatohepatitis, perpetuating hepatocellular injury and subsequent cell death, and promoting liver fibrosis.[7], [8], [9] However, the molecular basis behind the inflammatory response leading to steatohepatitis is still largely unknown.

Inflammatory caspases, including caspase-1, murine caspase-11, and human caspase-4/5, play important roles as mediators of inflammation.[10], [11], [12] Accumulating evidence has suggested that excessive activation of inflammatory caspases is implicated in the pathogenesis of hepatitis C viral infection, alcoholic liver disease and non-alcoholic steatohepatitis (NASH).13 Pyroptosis is the dominant response following the activation of inflammatory caspases, leading to pore formation in the plasma membrane, cell swelling, and massive release of the pro-inflammatory cellular contents.[12], [14], [15] Gasdermin D (GSDMD), a generic substrate for inflammatory caspases, has been shown to play a specific role in inflammatory caspase-mediated pyroptosis and also acts as a downstream effector of multiple inflammasomes.[16], [17] GSDMD exerts its pyroptosis executor function by releasing the cleaved gasdermin-N domain (GSDMD-N) that bears intrinsic pyroptosis-inducing activity and controls interleukin (IL)-1β release.[11], [18] As demonstrated by the crucial role of pyroptosis in immunity and disease, excessive uncontrolled pyroptosis may be detrimental to the host.[11], [12] Yet, despite these important functions, the potential effects of GSDMD and the mechanism of its action in steatohepatitis are still unknown. Moreover, no evidence is currently available on whether GSDMD-driven pyroptosis may represent a new avenue of therapeutic intervention for steatohepatitis. In this study, we investigated the significance of GSDMD in human and experimentally-induced NAFLD, particularly in steatohepatitis, and elucidated the pivotal role of GSDMD in steatohepatitis pathogenesis, by mediating lipogenesis and the amplification cascade of the nuclear factor-kappa B (NF-ĸB) signaling pathway. More importantly, we explored the clinical impact of GSDMD-N in patients with NASH, and demonstrated that GSDMD-N is positively correlated with the NAFLD activity score (NAS) and fibrosis, providing new insight into the potential treatment of NASH in humans.

Section snippets

Human samples

Human liver tissue samples of NAFLD/NASH were selected from bariatric surgery patients with no history of liver disease of other etiology from the Xijing Hospital, the Fourth Military Medical University. Histological assessments were determined by two pathologists in a double-blind manner.[19], [20] Samples from control individuals with normal liver histology were collected under percutaneous liver biopsy, from patients with no evidence of diabetes or hypertension. Patients with NAFLD were

Characteristics of the patient population

The main clinicopathological parameters of the patients evaluated in this study are described (Table S1). The histological characteristics of the patients are shown (Table S2). There were no statistically differences in age or gender between the three histological groups. Serum alkaline phosphatase (ALP), gamma glutamyl transpeptidase, and high-density lipoprotein cholesterol (HDL-C) levels did not differ statistically among the groups; whereas, serum alanine aminotransferase (ALT), aspartate

Discussion

The most important finding from this study is that GSDMD-induced pyroptosis was involved in human and murine steatohepatitis. Gsdmd−/− mice fed an MCD diet showed significantly reduced steatohepatitis compared with WT control mice fed with the same diet. Consistently, a noticeable improvement in liver inflammation, as well as a reduction in serum ALT levels and hepatic TG content, was observed in MCD-fed Gsdmd−/− mice. In addition, liver fibrosis was strongly attenuated in Gsdmd−/− mice after

Financial support

This work was supported by grants from National Natural Science Foundation of China (Nos. 81421003, 81730016, 81322037, 81572302, 81772650) and Independent Funds of the Key Laboratory (CBSKL2015Z12).

Conflict of interest

The authors have no conflicts to disclose.

Please refer to the accompanying ICMJE disclosure forms for further details.

Authors’ contributions

JL and KCW designed the experiments, supervised the study and revised the paper; FS commented on the study and provided the material support. BX, MZJ, YC and WJW completed the main experiments, wrote the first draft of the paper, prepared the figures and analyzed the data; DC and XWL helped with the main experiments; ZZ and DZ helped with the preparation of human samples; DMF and YZN helped with the experimental design and paper writing; All authors have reviewed the final version of the

Acknowledgements

We thank Professor Jun Yu, Xiang Zhang (Digestive Disease and The Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Hong Kong) for recommendations for animal models and experimental design. We thank Professor Gang Ji and Professor Jianyong Zheng (Xijing Hospital, The Fourth Military Medical University, Xi’an, China) for the material support. We thank members of the State Key Laboratory of Cancer Biology & Institute of Digestive Diseases for helpful discussions and

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    These authors contributed equally to this work.

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