The cross-talk of NLRP3 inflammasome activation and necroptotic hepatocyte death in acetaminophen-induced mice acute liver injury

Abstract

Overdose acetaminophen (APAP) can result in severe liver injury, which is responsible for nearly half of drug-induced liver injury in western countries. Previous studies have found that there existed massive hepatocellular necrosis and severe inflammatory response in APAP-induced liver injury. However, the mechanistic linkage between necroptosis and NLRP3 inflammasome pathway in APAP-induced hepatotoxicity remains poorly understood. In order to investigate the relationship between inflammation and hepatocytes death in APAP hepatotoxicity, a time-course model for APAP hepatotoxicity in C57/BL6 mice was established by intraperitoneal (i.p) injection of 300 mg/kg APAP in this study. The activity of serum enzymes and pathological changes of APAP-treated mice were evaluated, and the critical molecules in necroptosis and NF-κB-NLRP3 inflammasome signaling pathway were determined by immunoblot and immunofluorescence analysis. The results demonstrated that APAP overdose resulted in a severe liver injury. Furthermore, the expression of critical molecules in NLRP3 inflammasome and necroptosis pathways peaked at 12–24 h, and then was decreased gradually, which is consistent with the pattern of pathological injury induced by APAP. Our further investigation found that the level of IL-1β in mouse liver was closely correlated with the level of phosphorylated MLKL following exposure to APAP. Furthermore, inhibition of necroptosis with necrostatin-1 significantly suppressed the activation of NLRP3 inflammasome signaling. Taken together, our results highlighted that the cross-talk between necroptosis and NLRP3 inflammasome played a critical role for promoting APAP-induced liver injury. Inhibition of the interaction of inflammation and necroptosis by pharmaceutical methods may represent a promising therapeutic strategy for APAP-induced liver injury.

Keywords

Acetaminophen acute liver injury necroptosis NLRP3 inflammasome

Introduction

Acetaminophen (APAP), as an antipyretic and analgesic drug widely used in the world, is safe and effective at a therapeutic dose, but it can produces serious hepatotoxicity when overdose, resulting in severe acute hepatic injury and hepatic failure.¹ At present, the metabolism of APAP has been well characterized. N-acetyl-p-benzoquinone imine (NAPQI) is a highly reactive metabolite of APAP, which is believed to responsible for APAP hepatotoxicity. When therapeutic doses of APAP are ingested, the small amount of NAPQI is efficiently deactivated by conjugation with reduced glutathione (GSH), forming a mercapturic metabolite that is readily eliminated by the kidneys.² Following exposure to high-dose APAP, however, the endogenous glucuronide and sulfate cofactors become depleted, thus forming increased amounts of NAPQI. Excess NAPQI causes oxidative stress and binds covalently to liver proteins to form protein adducts, leading to the death of hepatocytes.^3,4

Although the precise mechanism by which APAP causes cellular injury is still unknown, it has been speculated that hepatocyte death and organ failure most likely result from the cumulative and additive effects of oxidative damage, mitochondrial dysfunction, and the disruption of Ca²⁺ homeostasis.³ Necrosis is one of the main forms of cell death, which is widely considered an unregulated form of cell death. However, this long-term view has been challenged by the discovery that tumor necrosis factor-α (TNF-α) can induce regulated necrotic cell death under apoptotic deficient conditions.⁵ Due to its regulatory properties, this necrotic form of cell death is called necroptosis.⁵ Different from typical necrosis, necroptosis is regulated by a series of signal transduction pathways. Among them, receptor interacting protein 1 (RIPK1), receptor interacting protein 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL) are the core components of the molecular mechanism of necroptosis.⁶ At present, necroptosis has been confirmed to exist in many chronic liver diseases such as viral hepatitis, autoimmune hepatitis, alcoholic liver diseases and non-alcoholic steatohepatitis.⁷ Recent studies have found that there existed a significant change in necroptosis-related proteins in APAP-treated mice livers. Furthermore, pharmacological and genetic inhibition of RIPK1 could significantly reduce the severity of APAP-induced l liver injury.^8

–11 These results have supported that RIPK-dependent necroptosis is involved in APAP-induced liver injury.

Inflammation in liver injury is commonly considered as a mechanism to remove debris and a stimulator to promote regeneration. However, the extensive and uncontrolled inflammatory response can further aggravate the liver damage.¹² APAP-induced acute hepatotoxicity demonstrates a necro-inflammatory injury pattern, sterile inflammation occurs in both mice and man after APAP overdose.¹³ Although the initial stage of toxicity is mediated by reactive metabolite formation and mitochondrial dysfunction, many studies in the last decade have suggested that a later stage of injury may at least partly through the recruitment of inflammatory leukocytes such as neutrophils and monocytes.^14,15 Necroptosis is believed as a programmed necrosis that induces cell death through the lysis of cells, resulting in the release of damage-associated molecular patterns (DAMPs) from the dead cells. DAMPs are potent inducers of inflammation, which was strongly correlated to the level of inflammation in the individual. The pyrin domain-containing protein 3 (NLRP3) inflammasome pathway has a pivotal role in the inflammatory-related disease pathogenesis, inducing IL-1β production and expression of the intracellular sensors. Pattern recognition receptors such as Toll-like receptors (TLRs) on macrophages recognize (DAMPs) and induce the production of pro-inflammatory cytokines and the liver recruitment of monocytes and neutrophils.¹⁶ APAP-induced acute liver injury is associated with the extensive inflammatory response and the infiltration of inflammatory cells, although it remains controversial whether the inflammation in livers would exacerbate damage.¹⁷ Recent reports suggest that RIPK3-MLKL necroptotic signaling can trigger activation of the NLRP3 inflammasome, resulting in IL-1β maturation and secretion.^18,19 Imaeda et al. found that NLRP3^−/− Casp1^−/−, and ASC^−/− mice were significantly less susceptible to APAP-induced liver injury than controls, that is, reduced mortality and liver injury in mice lacking components of the NLRP3 inflammasome.²⁰ In a recent study, we found that autophagy-activating agent rapamycin can significantly inhibit the activation of NLPR3 inflammasome and protect against APAP-induced liver damage, which further supports the critical role of NLPR3 inflammasome activation in APAP liver toxicity.²¹

Despite the critical role of necroptosis and NLRP3 inflammasome in APAP-induced liver injury, the time-course of them in the pathogenesis of APAP liver injury is still largely unknown. Especially, the causal relationship between necroptosis and inflammation signaling pathways in APAP-induced liver injury remains elusive. In the present study, we aim to observe the time-course of RIPK-mediated necroptosis and NLRP3 inflammasome signaling pathway, and explore the possible interaction between them and its contribution to APAP-induced hepatotoxicity.

Materials and methods

Materials

APAP (purity above 99%) and Necrostatin-1 were purchased from Sigma-Aldrich Chemical Co (St Louis, MO, USA). The kits for analysis of alanine/aspartate were obtained from Beijing Bioengineering Institute (Beijing, China). Protease Inhibitor Cocktail Tablets and Phosphatase Inhibitor Cocktail Tablets were purchased from Roche Applied Science and phosphatase Inhibitor Cocktail were purchased from Beyotime Biotechnology. Antibodies used in the study were IκBα (SC-1643, Lot# C1918), IKKα (SC-7606, Lot#H3018), IL-1β (SC-52012, Lot#A2518), NF-κB (SC-514451, lot#D1818) from Santa Cruz Biotechnology; MLKL (ab172868, lot#GR269926-11) p-MLKL (ser345, Lot#246882-30) are from Abcam Biotechnology; NLRP3 (D4D8T), p-IκBα (14D4, lot#18), RIPK1 (D94C12, lot#3) from cell signaling technology; RIPK3 (lot#8337-1701) from Novus biologicals; caspase-1 (lot#A27351509) from Adipogen life sciences. Monoclonal anti-β-actin (AC-15), peroxidase-conjugated Goat anti-Rabbit and Goat anti-Mouse IgG secondary antibodies, and Protease Inhibitor Cocktail were purchased from Sigma-Aldrich Chemical Co. BCA Protein assay Kit and SuperSignal® West Pico Chemiluminescent Substrate Kit were purchased from Pierce Biotechnology, Inc. (Rockford, IL, USA). Alexa Fluor 568 Goat Anti-Mouse IgG (H+L), Alexa Fluor® 488 Goat Anti-Rabbit IgG (H+L), and Hoechst 33343 are from life technologies (Carlsbad, CA, USA). All other chemicals were of analytical grade from commercial supplier.

Animals and treatments

Male C57/BL6 mice (20–22 g body weight) were purchased from Jinan Pengyue experimental animal company. All mice were housed in the SPF-class animal room with a 12 h light-dark cycle. Drinking water and complete-value mice feed were available ad libitum. In this study, 60 mice were randomly assigned to six groups (control, 3 h, 6 h, 12 h, 24 h, 48 h). The APAP solution was prepared in a volume of 50 g/ml using sodium chloride injection and dissolved in a 40°C water bath. After fasting 12 h, mice were injected intraperitoneally a dosage of 300 mg/kg while the control group was given sodium chloride injection alone, and then sacrificed at 0, 3, 6, 12, 24 and 48 h respectively. Furthermore, 40 mice were divided into four groups for Necrostatin-1 (NEC-1) intervention experiment (n = 10 mice/group): (1) control group, (2) 7 mg/kg NEC-1 group, (3) 300 mg/kg APAP group, (4) 300 mg/kg APAP + 7 mg/kg NEC-1 group; NEC-1 (7 mg/kg) was given intraperitoneally to animals of intervention groups 1 h prior to APAP administration. Then, all animals were sacrificed at the time-point of 24 h. The serum and liver tissue of all groups were collected for further analysis. All experiments were carried out in accordance with the NIH Guide for Care and Use of Laboratory Animals and followed the principles in the “Use of Animals in Toxicology.” All protocols were approved by the Institutional Animal Care and Use Committee of Shandong University.

Serum analyses and histological examination

The blood samples were kept at room temperature for 1 h. After centrifugation at 3000 × g for 15 min, serum was collected to measure the levels of ALT and AST with a Beckman AU480 automatic biochemical analyzer according to the manufacturer’s instructions. according to the manufacturer’s instructions. Furthermore, a segment of liver was fixed in 4% neutral buffered formalin solution at least 24 h. Then the liver tissues were dehydrated with a sequence of ethanol solutions, embedded in paraffin wax and sectioned at 4 µm thickness. At last, tissue sections were stained with hematoxylin and eosin (HE), and then observed to evaluate histopathological changes under light microscopy.

Tissue preparation and immunoblot analysis

Liver tissue was homogenized in RIPA buffer containing Protease Inhibitor Cocktail, phosphatase Inhibitor Cocktail, 50 mM Tris-HCL, 150 mM NaCl, 1% Triton-100, 1% sodium deoxycholate and 0.1%sodium dodecyl sulfate (SDS). Tissue homogenates were centrifuged at 12,000 × g for 15 min, and then the supernatants were kept for western analysis of target protein. Aliquots of tissue extracts in loading buffer were separated by electrophoresis on a 6% -12% sodium dodecyl sulfate (SDS) polyacrylamide gel. After the transfer of the resolved proteins to polyvinylidene fluoride (PVDF) membranes, the membranes were blocked with 2% non-fat milk in 0.1% Tween Tris-buffered saline (TBS-T) for 1 h at room temperature, and then incubated with primary antibody overnight at 4°C. The membranes were washed and incubated with peroxidase-conjugated secondary antibody for 1 h at room temperature. Subsequently, the membranes were detected by chemiluminescence kit. Finally, the immunoreactive bands of proteins were scanned with Agfa Duoscan T1200 scanner, and the digitize data were quantified as integrated optical density (IOD) using Image-Pro Plus software.

Immunofluorescence staining of livers

Paraffin sections of mice livers (5 μm) were firstly deparaffinized and hydrated. Then, antigen retrieval was performed by submerging the sections in citrate buffer and microwaving for 10 min. For immunostaining, the sections were treated with 10% serum, followed by overnight incubation with primary antibodies. The next day, sections were incubated with fluorescent secondary antibodies for 4 h at room temperature. Nuclei were stained with Hoechst 33343 and mounted with SlowFade Antifade Mountant. Finally, stained sections were observed and photographed with Nikon fluorescence microscope.

Statistical analysis

Experiment data were expressed as the mean ± SD. Statistical significance between experimental groups was assessed using one-way analysis of variance (ANOVA), followed by Dunnett’s post hoc tests. The relationship between p-MLKL and IL-1β was conducted by multiple correlation analysis, which was provided by SPSS23.0 statistical software, P value < 0.05 was used to indicate a statistically significant difference.

Results

APAP overdose induced severe liver injury in mice

Administration of a single dosage of APAP (300 mg/kg IP) to male C57BL/6 mice resulted in severe hepatic injury as measured by the serum enzyme activities. As shown in Figure 1A, serum ALT and AST levels in mice began to increase at 3 h after APAP treatment and reached the peak at the time point of 12 h and 24 h post-APAP. Then, the levels of both enzyme activity demonstrated a marked reduction at 48 h. Moreover, histopathological analysis showed that the liver exhibited various degrees of centrilobular hepatocyte necrosis accompanied with infiltration of inflammatory cells (Figure 1B). In keeping with the change of serum enzymes activity, the most severe centrilobular hepatocellular necrosis was primarily observed in the 12 h and 24 h groups. By contrast, the mice liver in the 48 h group exhibited a significant recovery.

Figure 1.

Biochemical evaluation and histopathological analysis of mice in the time-course model. Mice were intraperitoneally treated with APAP solution (300 mg/kg). Then the mice were sacrificed at a specified time point (0, 3, 6, 12, 24 and 48 h) after administration. Values are expressed as mean ± SD (n = 10). (A) Serum ALT and AST levels. (B) Histopathological analysis of representative HE stained liver samples from each group in the time-course model (100 × original magnification). *p < 0.05 compared to the control group.

The dynamic changes of RIPK1-RIPK3-MLKL axis in mice treated with APAP

To determine the role of RIPK-dependent necroptosis in APAP-induced hepatotoxicity, we have evaluated the level of RIPK1′RIPK3′MLKL and p-MLKL expression in mice liver by western blotting. As shown in Figure 2, although the expression of RIPK1 in mice livers was unchangeable following APAP exposure, an increased expression of RIPK3 was detected at 3 h post APAP treatment. and reached a maximum at 12–24 h, and then declined at 48 h post-APAP. Similarly, the level of p-MLKL was also significantly increased after APAP administration. These results

Figure 2.

Necroptosis signaling pathway in APAP-induced acute liver injury. The expression of RIPK1, RIPK3, MLKL, and p-MLKL was detected by Western blotting with β-actin as the loading control. The data of target proteins in the experiment groups were expressed as the mean percentage of the control (100%). The data are representative of three independent experiments and represented as mean ± SD. *p < 0.05 compared to the control group.

The activation of NF-κB-NLRP3 inflammasome pathway in mice treated with APAP

To study the changes of inflammatory-related signaling pathways in APAP liver injury, we firstly examined the critical proteins of NF-κB pathway in the liver of mice. As shown in Figure 3A, APAP administration resulted in a significant increase in IKK levels and an obvious nuclear translocation of NF-κB (p65), suggesting an activation of NF-κB signaling. Next, we further determined the activation of NLRP3 inflammasome. As demonstrated in Figure 3B, APAP exposure caused a significant increase in both NLRP3 and caspase-1 in mice liver. The level of caspase-1 and NLRP3 began to increase at 3 h and peaked at 12–24 h after APAP administration. Similarly, compared to the control animals, IL-1β production was also significantly increased in APAP-treated mice. Moreover, the cleaved caspase-1 and mature IL-1β were also dramatically elevated after the APAP treatment and peaked at 12–24 h, then decreased at 48 h. Collectively, our results supported that APAP treatment resulted in an activation of NF-κB-NLRP3 inflammasome pathway in mice liver.

Figure 3.

The activation of NF-κB-NLRP3 inflammasome signaling pathway in APAP-induced acute liver injury. (A) The alteration of NF-κB pathway in mice liver post APAP treatment; (B) the alteration of NLRP3 inflammasome in mice liver post APAP treatment. The level of target proteins was detected by Western blotting with β-actin or Lamin B as the loading control. The data of target proteins in the experiment groups were expressed as the mean percentage of the control (100%). The data are representative of three independent experiments and represented as mean ± SD. *p < 0.05 compared to the control group.

Relationship of hepatocyte necroptosis and inflammation in APAP-induced liver injury

In order to investigate the contribution of hepatocyte necroptosis and inflammation to APAP-induced liver injury, we have examined the relationship between MLKL and IL-1β content in mice livers by correlation analysis. As shown in Figure 4, a significant correlation was observed between MLKL and IL-1β in mice livers as the intoxication went on. Correlation coefficients of phosphorylated MLKL content with IL-1β of APAP-treated mice was 0.778 (P < 0.0001). Furthermore, we also analyzed the expression and localization of necroptosis and inflammatory molecules in mice liver by immunofluorescence. As expected, the co-localization of p-MLKL and IL-1β in the mice liver following APAP treatment significantly increased compared with the control (Figure 5). Remarkably, the most extensive co-localization of p-MLKL and IL-1β in the mice liver was observed at the timepoint of 12 h and 24 h post APAP treatment, which was consistent with the profile of necroptosis and inflammation in APAP hepatotoxicity.

Figure 4.

Pearson correlation analysis for p-MLKL and IL-1β in mice livers. The expression of p-MLKL and IL-1β was detected by Western blotting with β-actin as the loading control. The data of target proteins in the experiment groups were expressed as the percentage of the control (100%), n = 3.

Figure 5.

Co-localization of p-MLKL and IL-1β in mice liver sections by immunofluorescence analysis. p-MLKL, green; IL-1β, red; Hoechst 33343, blue. Scale bars = 50 μm.

Ncerostatin-1 inhibits NLRP3 inflammation signaling and protects against APAP-induced liver damage in mice

To further identify the effects of necroptosis on NLRP3 inflammation signaling, C57BL/6 mice were treated with 7 mg/kg Nec-1 prior to APAP administration and measured its effects. As an inhibitor of RIPK1, Nec-1 can efficiently inhibit necroptosis in in vitro and in vivo experiments. In the present study, pretreatment with Nec-1 almost completely inhibited the increase in ALT and AST activities and the hepatocytes necrosis. These results supported that Nec-1 can efficiently protect against APAP-induced liver injury (Figure 6A and 6B). More importantly, Nec-1 significantly suppresses the activation of NLRP3 signaling pathway. As showed in Figure 7, APAP-induced secretion of IL-1β and cleavage of caspase-1 in mice were markedly attenuated in mice pretreated with Nec-1, compared to those treated with APAP alone. Similarly, the enhancement of NLRP3 level at 24 h post-APAP was also almost completely inhibited by the pretreatment with Nec-1.

Figure 6.

Biochemical assessment, histopathological analysis of mice in intervention experiments with Nec-1. (A) Serum ALT and AST activities in Nec-1 intervention experiment. (B) Liver histopathological analysis of Nec-1 intervention experiment. Histopathological analysis of representative HE stained liver samples of each group in intervention experiment. Scale bar = 100 μm.

Figure 7.

Nec-1 treatment inhibited the activation of NLRP3 inflammasome signaling pathway in liver tissues of C57BL/6 mice following APAP administration. Immunoblot analysis for NLRP3, pro-caspase-1, pro-IL-1β, caspase-1, and IL-1β of NLRP3 inflammasome pathway in mice liver lysates. The data of target proteins in the experiment groups were normalized to β-actin, and then expressed as the mean percentage of the control (100%), n = 6. The data are representative of three independent experiments. *p < 0.05 compared to the control group, #p < 0.05 compared to APAP group.

Discussion

APAP can cause significant morbidity and mortality in cases of toxic-dose ingestion or improper use, which has been recognized as the most common cause of drug-induced liver injury in the United States and the western countries.²² Although APAP poisoning is generally considered to results in a centrilobular hepatocellular necrosis in humans and in mice, recent studies suggest that programmed cell death is involved in the process of APAP-induced liver cell death.^23,24 Moreover, there is severe inflammatory response in APAP-induced liver injury, but whether there is a second-stage injury due to inflammatory response in APAP-induced liver injury remains controversial.

In this study, the administration of 300 mg/kg APAP caused severe liver injury in mice, as indicated by the increased serum ALT and AST activities and extensive centrilobular necrosis. These changes were observed as early as 3 h post-APAP in mice, and peaked at 12–24 h. These results indicated that there existed a time-dependent profile in APAP-induced acute mice liver injury. The time-trend of ALT, AST were consistent with previous studies.^25,26 In order to explore the possible mechanistic linkage between inflammatory response and programmed cell death, we first examined the time-course of the necroptosis signaling pathway in APAP-induced mice liver injury. Necroptosis is a newly identified pathway of programmed cell death that can be induced by a variety of stimuli. Necroptosis is initiated when necroptotic stimuli sequentially activate the RIPK1, RIPK3, and MLKL protein through phosphorylation. Phosphorylated MLKL binds to and disrupts the plasma membrane of cells, resulting in cell lysis.^27,28 It has been reported that necroptosis might not contribute to APAP-induced necrosis.²⁹ However, in this study, we found that APAP treatment significantly increased RIPK3 and Phosphorylated MLKL in mouse livers, indicating an activation of necroptosis signaling pathway in APAP-treated mice. These results were in agreement with previous reports.^9
–11 In the meantime, our study also found that necroptosis was activated as early as 3 h after APAP exposure, which usually peaks at 12–24 h, and then decreases. This time-course is consistent with the profile of liver pathology and liver enzyme activity following APAP exposure. Therefore, it is reasonable to believe that necroptosis is involved in APAP-induced acute liver injury.

It is well-known that necroptosis involves cellular swelling and rupture, and has been considered to trigger inflammatory and immune responses. Intracellular components released from hepatocytes include nuclear DNA, mitochondrial DNA and proteins, they can act as the damage-associated molecular patterns (DAMPs) and activate the formation of the inflammasome complex in various cells such as Kupffer cells, thereby causing a release of cytokines and inflammatory mediators including IL-1β and TNF-α.^30,31 Furthermore, recent studies have demonstrated that active MLKL also triggered the assembly of NLRP3 inflammasome, which is required for the activity of IL-1β released during necroptosis.^18,32 As mentioned above, although the initial underlying mechanism of APAP-induced hepatotoxicity is the necrosis of hepatocytes, the second step in the liver injury is a sterile inflammation as a response to the necrotic hepatocytes. Although there are reports that the NLRP3 inflammasome does not play a relevant role in APAP hepatotoxicity.³³ But recently, NLRP3 inflammasome has been identified as a potential mediator in the mouse model of APAP overdose, which plays a crucial role in the second step of proinflammatory cytokine activation following APAP-induced liver injury.^20,34

The activation process of inflammasome is considered to be a two-step process, the first step is the up-regulation of inflammasome expression, the second step is the activation of functional inflammasome.³⁵ NF-κB is extensively involved in the inflammatory process and mediates the transcriptional and translational upregulation of NLRP3 and inflammatory cytokines including pro-IL-1β and pro-IL-18.³⁶ Furthermore, NLRP3 inflammasome can be activated by a wide spectrum of PAMPs and sterile DAMPs. NLRP3 inflammasome complex further activates the protease caspase-1, which cleaves pro-IL-1β and pro-IL-18, converting them to their active forms. In the present study, APAP treatment caused a significant increase in IKK levels and an obvious nuclear translocation of NF-κB (p65), suggesting an activation of NF-κB signaling. In the meantime, marked induction of NLRP3, capsase-1 and IL-1β was also observed in mice liver, which was in accordance with the previous reports.^20,21 Furthermore, the activated caspase-1 and mature forms of IL-1β also increased significantly, which confirmed the activation of NLRP3 inflammasome following APAP. Noteworthily, the enhancement in these proteins mentioned-above initially appeared 3 h after APAP exposure, usually peaked at 12–24 h, and then gradually decreased, which was similar to the time-course of the necroptosis-related proteins in mice liver following APAP.

Although NLRP3 inflammasome activation is known to play a pathological role in APAP-induced liver injury, its exact role in mediating necroptosis-dependent hepatocyte death still remains not well understood. In this study, a markedly increase in the co-localization of IL-1β and p-MLKL was observed in mice livers after APAP treatment. Furthermore, we have also studied their correlation in the pathogenesis of APAP-induced acute liver injury, and found that the activation of NLRP3 inflammasome in mouse liver was correlated with the phosphorylation of MLKL and up-regulation of the necroptotic signaling pathway.

Although the time course of APAP-induced hepatotoxicity as well as the role of inflammasome in the process have been extensively investigated, the relationship between necroptosis and inflammasome remains largely unknown.^25,37,38 To further testify the mechanistic linkage between necroptosis and NLRP3 inflammasome in APAP liver injury, we next examined whether the necroptosis-inhibiting drug could reduce the upregulation of NLRP3 inflammasome induced by APAP. As an inhibitor of RIPK1, necrostatin-1 (Nec-1) was extensively used to inhibit necroptosis in in vitro and in vivo experiments.³⁹ As expected, necrostatin-1 pretreatment almost completely blocked APAP-induced liver injury, as indicated by pathological analysis of mouse liver sections. The phenomenon that Nec-1 efficacy against APAP-induced liver injury has also partially confirmed in other experiments.¹⁰ In the meantime, Nec-1 pretreatment markedly suppressed the activation of NLRP3 inflammasome and attenuated APAP-induced caspase-1 activation and IL-1β production. In a recent study, blockade of either RIPK1 or RIPK3 with pharmacological and genetic methods was associated with the lower caspase-1 activation in APAP-mediated liver injury.⁹ Due to the critical role of caspase-1 in NLRP3 inflammasome activation, the data in Deutsch’ s paper were in agreement with our results. Taken together, our results have supported that there existed a causal linkage between the necroptosis and NLRP3 signaling in the pathogenesis of APAP-induced acute liver injury.

Mechanistically, we speculated that necroptosis could form a forward feedback loop with NLRP3 inflammasome in the pathogenesis of APAP hepatotoxicity. On one hand, necroptosis enhances NLRP3 inflammasome signaling by directly releasing the intracellular components as DAMPs and leads to the prolonged activation of NF-κB-NLRP3 inflammasome signaling; on the other hand, the activated NLRP3 inflammasome induces the synthesis, maturation and secretion of inflammatory factors, thus promoting necroptosis in a positive feedback manner and further aggravating liver injury. In this respect, the inhibition of necroptosis, either genetically or with small-molecule inhibitors, has also been reported to lessen disease severity in several mouse models.^6,40 Additionally, recent studies have also found that RIPK1 exist in two main forms of “closed-conformation” and “open-conformation.” The open-conformation and kinase-active RIPK1 initiates apoptosis and necroptosis. By contrast, the closed-conformation form of RIPK1 can directly activate the NF-κB signaling pathway regardless of its defective kinase activity.⁴¹ In APAP-induced hepatotoxicity, whether RIPK1 can directly activate the NF-κB signaling pathway needs further research.

In conclusion, our work highlights the cross-talk between necroptosis and NLRP3 inflammasome is important for promoting APAP-induced liver injury. The finding in this study implies that the inhibition of necroptosis by RIPK inhibitors not only alleviate the hepatocyte necroptotic death, may also repress NLRP3 inflammasome signaling, leading to the attenuation of inflammatory response. The attenuation of both necroptotic and inflammatory signaling pathways could protect against APAP-induced liver injury.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by Key research and development plan of Shandong Province (2018GSF118013) and National Natural Science Foundation of China (No. 81673209).

ORCID iD

L Li

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