Hepatocyte cell death, inflammation and oxidative stress constitute key pathogenic mechanisms underlying non-alcoholic fatty liver disease (NAFLD). We aimed to investigate the role of necroptosis in human and experimental NAFLD and its association with tumour necrosis factor α (TNF-α) and oxidative stress. Serum markers of necrosis, liver receptor-interacting protein 3 (RIP3) and phosphorylated mixed lineage kinase domain-like (MLKL) were evaluated in control individuals and patients with NAFLD. C57BL/6 wild-type (WT) or RIP3-deficient (RIP3−/−) mice were fed a high-fat choline-deficient (HFCD) or methionine and choline-deficient (MCD) diet, with subsequent histological and biochemical analysis of hepatic damage. In primary murine hepatocytes, necroptosis and oxidative stress were also assessed after necrostatin-1 (Nec-1) treatment or RIP3 silencing. We show that circulating markers of necrosis and TNF-α, as well as liver RIP3 and MLKL phosphorylation were increased in NAFLD. Likewise, RIP3 and MLKL protein levels and TNF-α expression were increased in the liver of HFCD and MCD diet-fed mice. Moreover, RIP3 and MLKL sequestration in the insoluble protein fraction of NASH (non-alcoholic steatohepatitis) mice liver lysates represented an early event during stetatohepatitis progression. Functional studies in primary murine hepatocytes established the association between TNF-α-induced RIP3 expression, activation of necroptosis and oxidative stress. Strikingly, RIP3 deficiency attenuated MCD diet-induced liver injury, steatosis, inflammation, fibrosis and oxidative stress. In conclusion, necroptosis is increased in the liver of NAFLD patients and in experimental models of NASH. Further, TNF-α triggers RIP3-dependent oxidative stress during hepatocyte necroptosis. As such, targeting necroptosis appears to arrest or at least impair NAFLD progression.

CLINICAL PERSPECTIVES

  • Regulated necrosis or necroptosis, an immunogenic programmed cell death type, morphologically similar to necrosis, has recently been implicated in the pathogenesis of inflammation-driven liver disease. In turn, necroinflammation is an important pathological feature of NASH, whereas TNF-α is a well-established trigger of necroptosis, involved in NASH pathogenesis.

  • In the present study, we found that liver RIP3 levels are significantly increased in several chronic liver diseases, correlating with steatohepatitis histological severity. In addition, necroptosis, as defined by RIP3-dependent MLKL activation, is triggered in the liver of mouse models of NASH and in human NAFLD, whereas absence of RIP3 ameliorates liver injury, steatosis, inflammation and fibrosis in MCD-induced experimental NASH. Mechanistically, TNF-α-induced necroptosis of primary murine hepatocytes involves RIP3-dependent ROS production.

  • Targeting necroptosis may provide an unprecedented opportunity to develop novel therapeutic strategies to attenuate or prevent liver injury, inflammation and fibrosis associated with NAFLD pathogenesis.

INTRODUCTION

Necrosis is considered an accidental process characterized by cell swelling and early loss of plasma membrane integrity, with consequent leakage of pro-inflammatory mediators. The unregulated nature of necrosis precludes the design of therapeutic interventions for protection of necrosis-associated cell injury and inflammation. Interestingly, regulated necrosis or necroptosis was recently described as a novel cell death pathway, morphologically similar to that of necrosis, but occurring in a programmed manner, through well-defined biochemical pathways [1].

Necroptosis may occur in response to multiple stimuli, tumour necrosis factor-α (TNF-α) being the most well-studied initiation signal. Typically, TNF-α-dependent necroptosis is initiated by interaction between receptor-interacting proteins 1 (RIP1) and 3 (RIP3), which originate in the necrosome, an oligomeric amyloid signalling complex. At the functional level, the auto- and trans-phosphorylation of RIP1 and RIP3 are required for necrosome assembly and activation of necroptotic signalling [2]. In that regard, necrostatin-1 (Nec-1), a tryptophan-based molecule that allosterically inhibits RIP1 kinase activity, blocks necroptosis [3]. However, in specific cell contexts, increased RIP3 levels [46] or overexpression of a RIP3 phospo-mimetic mutant [7] can trigger necroptosis in the absence of RIP1, suggesting that RIP3 is a unique regulator of necroptotic cell fate. RIP3 recruits and phosphorylates the pseudokinase mixed lineage kinase domain-like (MLKL), which in turn oligomerizes and causes irreversible cellular membrane damage, resulting in necrotic cell death [8]. In addition, overproduction of reactive oxygen species (ROS) has been described as a possible contributing factor in some cellular contexts [3,4,9].

The physiological relevance of necroptosis has been demonstrated in a variety of paradigms. In particular, necroptosis is involved in the pathogenesis of several inflammatory disorders, including pancreatitis [10] and chronic inflammation of gut [11,12] and skin [13]. Likewise, necroptosis is arising as a likely pathological feature of inflammation-driven liver diseases. For instance, RIP3 mediates ethanol or drug-induced liver injury in vivo [8,14,15], whereas apoptosis and RIP3-dependent necroptosis are simultaneously activated in an animal model of chronic hepatic inflammation [16]. In addition, concanavalin A-induced hepatic failure is associated with hepatocyte necroptosis and aberrant TNF-α signalling [17,18]. Curiously, as a potent proinflammatory cytokine, TNF-α is involved in the pathogenesis of a broad range of liver diseases, including viral hepatitis, alcoholic hepatitis, acute liver failure and non-alcoholic fatty liver disease (NAFLD) [19]. NAFLD is the most common chronic liver disease and its prevalence and incidence is increasing due to strong association with obesity and the metabolic syndrome. NAFLD encompasses a spectrum of liver dysfunction ranging from simple fatty liver or hepatic steatosis to hepatic necrosis and inflammation, characteristic of non-alcoholic steatohepatitis (NASH). Further, NAFLD may potentially progress to severe long-term consequences, including cirrhosis, hepatocellular carcinoma and, ultimately, premature death. Whereas NAFLD triggering is associated with lipid deposition within hepatocytes, the subsequent mechanisms of liver damage are multifactorial and relate to metabolic changes, oxidative stress, miRNA expression and cytokine production [20]. Their exact contribution and interplay during NAFLD progression remains far from completely understood.

We hypothesize that necroptosis represents a major pathway mediating the pathogenesis of inflammation-driven liver diseases. This prompted us to determine the involvement of necroptosis in NAFLD pathogenesis in humans and in experimental murine models of hepatic steatosis and NASH. Further, we aimed at establishing the involvement of TNF-α and oxidative stress in necroptotic signalling in hepatocytes.

METHODS

Patients, histological findings and grading

NAFLD liver specimens were obtained from morbidly obese patients undergoing bariatric surgery, as previously described [21]. Moreover, liver tissue was also prospectively and sequentially collected from patients that fulfilled the clinical and pathological diagnostic features of alcoholic steatohepatitis (ASH; n=5), hepatitis B (n=5) and hepatitis C (n=4). Control liver specimens were obtained from five individuals who were potential liver donors, with normal liver histology and biochemistry. No statistical differences were observed in age and gender between patient groups. All liver specimens were processed conventionally for diagnostic purposes and were blindly evaluated by an experienced pathologist.

Informed written consent was obtained from all patients and the study protocol conformed to the Ethical Guidelines of the 1975 Declaration of Helsinki, as reflected in a priori approval by the Hospital of Santa Maria (Lisbon, Portugal) Human Ethics Committee.

Paraffin-embedded liver tissue sections from ASH and NAFLD patients were stained with Haematoxylin and Eosin (H&E). The Gordon and Sweet's Silver Staining method was used for identification of reticular fibres, Chromotrope-Aniline Blue (CAB) for connective tissue and Perl's Prussian Blue for iron. Steatosis was graded from 0 to 3 based on the percentage of steatotic hepatocytes (0, none; 1, <33%; 2, 33%–66%; 3, >66%). In addition, portal and lobular inflammation and portal and lobular fibrosis were semi-quantitatively graded on a scale of 0–4 (0, absence; 1, mild; 2, moderate; 3, severe degree and 4, cirrhosis), as previously reported [22]. Eleven patients were classified as having NASH and five as having simple steatosis. In control individuals (n=5), all parameters were graded 0.

Serum analyses

Serum samples from a cohort of morbidly obese patients with clinical and biopsy-proven diagnosis of steatosis (n=29) and NASH (n=15) were used for biochemical assays. Serum of liver disease-free individuals was also analysed (n=5). Routine laboratory assays included total cholesterol, high-density lipoprotein (HDL)-cholesterol, low-density lipoprotein (LDL)-cholesterol, triacylglycerol, aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transpeptidase (γ-GT), total bilirubin, fasting serum glucose and fasting insulin, using the standard techniques of clinical chemistry laboratories. In addition, sandwich ELISA was used to determine cytokeratin 18 full-length (CK18-M65; 10020, Peviva), high-mobility group box 1 (HMGB1; ST51011, IBL International GmbH), cyclophilin A (CypA; E01C0613, BlueGene Biotech) and TNF-α (HSTA00D, R&D System Inc.) serum levels. In addition, phospho-MLKL (p-MLKL), total MLKL and RIP3 levels were analysed in the liver specimens of these patients by immunoblotting. Demographic, clinical and routine laboratory data of a cohort of patients at different biopsy-proven NAFLD stages are summarized in Table 1. 

Table 1
Demographic, clinical and laboratory data of NAFLD patients.

Data are presented as mean ± S.E.M. No statistical significant differences were found between groups. In healthy volunteers, all biochemical parameters were within the reference values.

Steatosis (n=29)NASH (n=15)
Age (years) 42±2 41±3 
Gender (M/F) 10/19 6/9 
Body mass index (kg/m244.9±7.9 47.9±7.1 
Total cholesterol (mmol/l) 4.9±0.2 5.3±0.3 
HDL-cholesterol (mmol/l) 1.9±0.1 1.2±0.1 
LDL-cholesterol (mmol/l) 3.1±0.2 3.3±0.3 
Triacylglycerol (mmol/l) 1.4±0.1 1.6±0.2 
Fasting glucose (mmol/l) 5.3±0.2 5.2±0.2 
Fasting insulin (pmol/l) 136.1±13 137.0±14 
Total bilirubin (mmol/l) 12.4±1.3 11.1±3.0 
Steatosis (n=29)NASH (n=15)
Age (years) 42±2 41±3 
Gender (M/F) 10/19 6/9 
Body mass index (kg/m244.9±7.9 47.9±7.1 
Total cholesterol (mmol/l) 4.9±0.2 5.3±0.3 
HDL-cholesterol (mmol/l) 1.9±0.1 1.2±0.1 
LDL-cholesterol (mmol/l) 3.1±0.2 3.3±0.3 
Triacylglycerol (mmol/l) 1.4±0.1 1.6±0.2 
Fasting glucose (mmol/l) 5.3±0.2 5.2±0.2 
Fasting insulin (pmol/l) 136.1±13 137.0±14 
Total bilirubin (mmol/l) 12.4±1.3 11.1±3.0 

Animals and diets

Male C57BL/6 6-week-old mice (Harlan Laboratories) were fed either standard chow diet (control; n=5; Mucedola) or high-fat (35% total fat, 54% trans-fatty acid enriched) choline-deficient diet (HFCD; n=4–5; Harlan Laboratories) for 6 and 18 weeks. Alternatively, 5-month-old C57BL/6 wild-type (WT) or RIP3-deficient (RIP3−/−) mice were fed either a chow diet or a methionine and choline-deficient (MCD; n=5) diet (TestDiet) for 2 and 8 weeks. At the indicated time-points, animals were killed by exsanguination under isoflurane anaesthesia. The liver was removed; one lobe was collected, rinsed in normal saline and immediately flash-frozen in liquid nitrogen for protein and RNA extraction; another lobe was included in optimal cutting temperature compound (4583, Tissue Tek) for histochemistry of fat by Oil Red O staining (O-0625, Sigma–Aldrich Co.). Paraffin-embedded sections (3–4 μm) were stained with H&E, CAB or Masson's Trichrome. Liver sections were scored in a blinded fashion by experienced pathologists, using a four-point severity scale (0, normal; 1, mild; 2, moderate; 3, severe), for steatosis, inflammatory cell infiltration and fibrosis. In the MCD model, serum was also collected and ALT and AST determined using standard clinical chemistry techniques. All procedures were reviewed and approved by the local ethics committee and national competent authorities for animal protection. Animals received humane care in a temperature-controlled environment with a 12-h light–dark cycle, complying with the Institute's guidelines and as outlined in the ‘Guide for the Care and Use of Laboratory Animals’ prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86-23 revised 1985).

Quantitative RT-PCR

RNA was extracted from animal liver samples using the TRIzol reagent according to the manufacturer's instructions (Life Technologies Corp.). Real-time RT-PCR was performed in an Applied Biosystems 7300 System (Life Technologies Corp.). The following primer sequences were used: for the TNF-α gene, 5′-AGG CAC TCC CCC AAA AGA TG-3′ (forward) and 5′-TGA GGG TCT GGG CCA TAG AA-3′ (reverse); for the interleukin (IL)-1β gene, 5′-TGC CAC CTT TTG ACA GTG ATG-3′ (forward) and 5′-TGA TGT GCT GCT GCG AGA TT-3′ (reverse); for the collagen-1-α1 gene, 5′-CTG ACT GGA AGA GCG GAG AG-3′ (forward) and 5′-GAC GGC TGA GTA GGG AAC AC-3′ (reverse); for transforming growth factor (TGF)β gene, 5′-CTG CTG ACC CCC ACT GAT AC-3′ (forward) and 5′GTG AGC GCT GAA TCG AAA GC-3′ (reverse); and for the hypoxanthine phosphoribosyltransferase (HPRT) gene, 5′-GGT GAA AAG GAC CTC TCG AAG TG-3′ (forward) and 5′-ATA GTC AAG GGC ATA TCC AAC AAC A-3′ (reverse). Two independent reactions for each primer set were assessed in a total volume of 12.5 μl containing 2× Power SYBR green PCR master mix (Thermo Fisher Scientific) and 0.6 μM (each) primer. The relative amounts of TNF-α, IL-1β, collagen-1-α1, TGFβ and HPRT were calculated based on the standard curve. TNF-α, IL-1β, collagen-1-α1 and TGFβ mRNA levels were normalized to the level of HPRT and expressed as fold change from controls.

Cell culture and treatments

Primary rat hepatocytes were isolated from male rats (100–150 g) by collagenase perfusion, as previously described [23,24]. Primary mouse hepatocytes were isolated from male WT and RIP3−/− mice using liver perfusion and liver digest medium (Gibco, Life Technologies Corp.) according to the manufacturer's protocols with some modifications. Briefly, mice were killed with isofluorane overdose, inferior vena cava cannulated and the liver perfused in situ with liver perfusion medium (37°C), followed by perfusion with liver digest medium (pH 7.4, 37°C). The liver was removed and then gently minced in 20 ml of liver digest medium. The liver cell suspension was homogenized with 20 ml of Williams-E Glutamax Complete Medium (Gibco) containing 10% FBS (Gibco) and then filtered through a 70 μm cell strainer. Cell suspension was centrifuged at 50 g for 5 min. The pellet was resuspended in 10 ml of medium and added to 10 ml of buffered Percoll, containing 9 ml of Percoll (Sigma–Aldrich Co.) plus 1 ml 10× PBS (Life Technologies Corp.). Cell suspension was centrifuged at 50 g for 5 min and the pellet resuspended in Williams-E Glutamax Complete Medium containing penicillin (100 units/ml)/streptomycin (100 μg/ml; Gibco) and 4% FBS. Cell viability, as determined by Trypan Blue exclusion, was generally >85%. After isolation, primary murine hepatocytes were plated on Primaria™ tissue culture dishes (BD Biosciences) at 5 × 104 cells/cm2. Cells were maintained at 37°C in a humidified atmosphere of 5% CO2.

Primary murine hepatocytes were pre-treated with 50 μM of a pan-caspase inhibitor, zVAD-fmk (Enzo Life Sciences Inc.) and/or 100 μM Nec-1 (Sigma–Aldrich Co.) or DMSO (Sigma–Aldrich Co.) vehicle control (final concentration of 0.1%). After 1 h, cells were exposed to 400 μM palmitic acid (PA; Sigma–Aldrich Co.) or TNF-α (10 ng/ml; PeproTech EC Ltd.) plus cycloheximide (CHX; 0.5 μg/ml; Sigma–Aldrich Co.) or vehicle control. PA was prepared in isopropyl alcohol at a stock concentration of 80 mM. PA was added to Complete William's E medium containing 1% BSA to ensure a physiologic ratio between bound and unbound PA in the medium, approximating the molar ratio present in the plasma [25]. The concentration of PA used in the experiments was less than the fasting total non-esterified fatty acid plasma concentrations observed in human NASH [26,27]. The concentration of isopropyl alcohol was 0.5% in final incubations; this concentration was used as vehicle control in PA experiments. After 24 h, hepatocytes were processed for cell death assays, caspase-3/7 activity measurements and immunocytochemistry analyses.

For functional analyses, primary rat hepatocytes were transfected at the moment of plating with 100 pM of a siRNA nt against ripk3 (siRIPK3; s140868, Ambion, Life Technologies Corp.) or with a siRNA control, using Lipofectamine™2000 (Invitrogen, Life Technologies Corp.), according to the manufacturer's instructions. Primary rat hepatocytes were harvested at 48 h post-transfection for protein extraction, to confirm RIP3 silencing. Alternatively, primary rat and mouse hepatocytes were pre-treated for 1 h with zVAD-fmk and/or Nec-1 before incubation with TNF/CHX. After 3 and 24 h, cells were harvested for total ROS levels detection and cell-death assays, respectively.

Total and soluble/insoluble protein extraction

For isolation of total protein extracts, liver pieces and primary rat hepatocytes were homogenized using a glass dounce homogenizer in ice-cold lysis buffer (10  mM Tris/HCl, pH 7.6, 5  mM MgCl2, 1.5  mM potassium acetate, 1% Nonidet P-40, 2  mM DTT) and 1× Halt Protease and Phosphatase Inhibitor Cocktail (Pierce, Thermo Fisher Scientific). The lysate was centrifuged at 3200 g for 10 min at 4°C and the supernatant recovered and stored at −80°C. For soluble/insoluble protein extraction, livers were homogenized using a glass dounce homogenizer in radio-immunoprecipitation assay (RIPA) buffer (50 mM Tris/HCl, pH 8; 150 mM NaCl; 1% NP-40; 0.5% sodium deoxycholate; 0.1% SDS) and 1× Halt Protease and Phosphatase Inhibitor Cocktail (Pierce). The suspension was centrifuged at 15000 g for 20 min at 4°C. The resultant supernatant was collected as the soluble protein fraction and was centrifuged a second time for complete removal of all insoluble proteins. Insoluble protein fractions contained in pellets were resuspended in RIPA buffer supplemented with 8 M urea and were sonicated at 30%–40% power for 7 s. Protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad Laboratories), according to the manufacturer's specifications.

Immunoblotting

Steady-state levels of RIP3 and MLKL were determined by immunoblot analysis. Briefly, 60 μg of total protein extracts, insoluble or soluble protein fractions were separated on an 8% SDS/PAGE. Following electrophoretic transfer on to nitrocellulose membranes and blocking with 5% milk solution, blots were incubated overnight at 4°C with primary rabbit polyclonal antibodies against RIP3 (1:200, Santa Cruz Biotechnology Inc.), mouse MLKL (1:500, SAB1302339, Sigma–Aldrich), human p-MLKL (1:1000, Abcam plc) and human MLKL (1:1000, Abcam plc) and with a secondary antibody conjugated with horseradish peroxidase (Bio-Rad Laboratories) for 3 h at room temperature. Membranes were processed for protein detection using Super Signal substrate (Pierce). β-Actin (1:20000; Sigma–Aldrich) and Ponceau S staining (Merck) were used as loading controls. We have previously validated Ponceau S staining as a loading control for immunoblot analysis [28], which is particularly relevant when regular housekeeping proteins are inadequate controls, like for mouse liver insoluble/soluble protein fractions.

Cell death and caspase activity measurements

General cell death was evaluated using the lactate dehydrogenase (LDH) Cytotoxicity Detection KitPLUS (Roche Diagnostics GmbH), following the manufacturer's instructions. In each experiment set-up, experimental LDH values were normalized with maximum releasable LDH activity in the cells, after cell disruption with the provided lysis solution. Hoechst 33258 (Sigma–Aldrich Co.) labelling of attached cells was used to detect apoptotic nuclei by morphological analysis, as previously described [29]. Caspase-3/-7 activity was measured using the Caspase-Glo 3/7 Assay (Promega Corp.), according to the manufacturer's protocol.

Analysis of total ROS levels

ROS levels were analysed through the use of 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA; Sigma–Aldrich Co.), a cell-permeant non-fluorescent molecule that is oxidized by ROS to form dichlorofluorescein, a fluorescent compound. Primary murine hepatocytes were incubated with 10 μM H2DCFDA at 37°C for 30 min. Cells were then washed with PBS (Life Technologies Corp.). Twenty-five milligrams of liver tissue was homogenized using a glass dounce in 500 μl of ice-cold PBS. To remove insoluble particles, the lysate was centrifuged at 10200 g for 5 min at 4°C and the supernatant was recovered. Fifty microlitres of the lysate were incubated with 10 μM H2DCFDA at room temperature for 30 min. The emission of green fluorescence was measured using the GloMax-Multi+ Detection System (Promega Corp.). Fluorescence values were corrected with total protein content.

Immunochemistry and image analysis

RIP3 expression was evaluated by immunohistochemistry in human liver tissue. Paraffin-embedded liver sections were deparaffined, rehydrated and boiled three times in 10 mM citrate buffer, pH 6. Sections were then incubated for 1 h in blocking buffer, containing 0.3% Triton X-100 (Sigma–Aldrich Co.), 1% FBS (Life Technologies Corp.) and 10% normal donkey serum (Jackson ImmunoResearch Laboratories). RIP3 expression and localization in primary rat hepatocytes was evaluated by immunocytochemistry. Cells were washed twice, fixed with paraformaldehyde in PBS (4%, w/v) and then blocked for 1 h at room temperature in PBS, containing 0.1% Triton X-100, 1% FBS and 10% normal donkey serum. For both staining procedures, samples were then incubated with a primary antibody reactive to RIP3 (1:50; Santa Cruz Biotechnology) overnight at 4°C. After rinsing, the primary antibody was developed by incubating with a 1:200 secondary DyLight 594-conjugated anti-rabbit antibody (Jackson ImmunoResearch Laboratories) for 2 h at room temperature. Hepatocyte nuclei were stained with Hoechst 33258 at 50 μg/ml in PBS for 6 min at room temperature. Samples were mounted using Fluoromount-G (Beckman Coulter). Detection of RIP3 in cells was visualized using an AxioScope.A1 microscope (Carl Zeiss Microscopy GmbH). Images were acquired, under 400× magnification, using an AxioCam HRm camera with the AxioVision software (release 4.8; Carl Zeiss Microscopy GmbH). Semi-quantitative analysis of mean fluorescence intensities of RIP3 was performed using the NIH ImageJ software. Eight images per sample were obtained. Images were converted into an 8-bit format and the background was subtracted. An intensity threshold was set and kept constant for all images analysed. RIP3 fluorescence intensity in primary rat hepatocytes was normalized with the number of cells per microscopic field.

Densitometry and statistical analysis

The relative intensities of protein bands were analysed using the Image Lab densitometric analysis program (version 5.1; Bio-Rad Laboratories). Results from different groups were compared using the Student's t test or Kruskal–Wallis non-parametric ANOVA. Values of P<0.05 were considered statistically significant. All statistical analysis was performed with GraphPad Prism 5 software (GraphPad Software).

RESULTS

RIP3 is increased in the liver of patients with chronic liver disease

Fatty acids, alcohol, viruses and the immune response are well-known triggers of liver injury and chronic disease, at least in part by strongly activating liver cell death [1]. Nevertheless, the relative contribution of necroptosis to the pathogenesis of inflammation-associated human liver pathologies, namely NAFLD, ASH, hepatitis B and hepatitis C (Figure 1A), has not been systematically explored.

RIP3 expression is increased in patients with chronic liver disease

Figure 1
RIP3 expression is increased in patients with chronic liver disease

(A) H&E staining of representative liver sections of normal controls (n=5), patients with steatosis (n=5), NASH (n=11), ASH (n=5), hepatitis B (Hep B; n=4) and hepatitis C (Hep C; n=5). Scale bar=100 μm. (B) Representative RIP3 immunostaining (red) in liver tissue from patients. Nuclei were counterstained with Hoechst 33258 (blue). The corresponding histogram shows the quantification of RIP3 mean fluorescence intensity as described in ‘Materials and Methods’. Values are from at least eight images per liver sample. Scale bar=10 μm. Data are expressed as mean ± S.E.M. fold change. §P<0.05 and *P<0.01 compared with Control.

Figure 1
RIP3 expression is increased in patients with chronic liver disease

(A) H&E staining of representative liver sections of normal controls (n=5), patients with steatosis (n=5), NASH (n=11), ASH (n=5), hepatitis B (Hep B; n=4) and hepatitis C (Hep C; n=5). Scale bar=100 μm. (B) Representative RIP3 immunostaining (red) in liver tissue from patients. Nuclei were counterstained with Hoechst 33258 (blue). The corresponding histogram shows the quantification of RIP3 mean fluorescence intensity as described in ‘Materials and Methods’. Values are from at least eight images per liver sample. Scale bar=10 μm. Data are expressed as mean ± S.E.M. fold change. §P<0.05 and *P<0.01 compared with Control.

RIP3 is a crucial player in the necroptotic signalling pathway. For instance, cell death shifts from apoptosis to necroptosis when RIP3 is overexpressed in NIH3T3 mouse fibroblasts [4]. Moreover, the expression of RIP3 in different cell types correlates with their responsiveness to induction of necroptosis [10]. Thus, to assess the presence of necroptosis in chronic liver diseases, RIP3 protein expression was evaluated by immunohistochemistry in liver biopsies from patients with steatosis, NASH, ASH, hepatitis B and hepatitis C, as well as in histologically normal subjects (Figure 1B). In normal livers, RIP3 was weakly expressed. Notably, all chronic liver disease patients showed a significant increase in liver RIP3 expression, when compared with healthy controls (at least, P<0.05), except for steatosis. Of note, hepatic steatosis and steatohepatitis grading and severity (Table 2) correlated with induction of RIP3. Particularly, a positive correlation was found between RIP3 expression and the relative degree of portal fibrosis in a cohort of control individuals, steatosis, NASH and ASH patients (r2=0.91, P<0.05).

Table 2
Histological data of steatosis, NASH and ASH patients

Histological features are presented as number and percentage of cases in each grade. Grade 4 fibrosis is equivalent to cirrhosis. All parameters were graded 0 in control individuals. Kruskal–Wallis non-parametric ANOVA was performed to assess differences between patient groups. For lobular inflammation, P-value is at least < 0.05 in ASH or NASH compared with steatosis. *200× magnification. ns, not significant.

Steatosis (n=5)NASH (n=11)ASH (n=5)NASH compared with ControlASH compared with Control
Steatosis      
 0 (none)  1 (9%) 1 (20%) P<0.01 ns 
 1 (mild) 3 (60%) 3 (27%) 2 (40%)   
 2 (moderate) 1 (20%) 3 (27%) 1 (20%)   
 3 (severe) 1 (20%) 4 (36%) 1 (20%)   
Lobular inflammation      
 0 (none) 5 (100%)   P<0.01 P<0.05 
 1 (<2)*  1 (9%) 2 (40%)   
 2 (2–4)*  8 (73%) 2 (40%)   
 3 (>4)*  2 (18%) 1 (20%)   
Portal inflammation      
 0 (none)  2 (18%)  P<0.05 P<0.01 
 1 (mild) 5 (100%) 9 (82%) 4 (80%)   
 2 (moderate)      
 3 (severe)   1 (20%)   
Lobular fibrosis      
 0 (none)  1 (9%)  P<0.05 P<0.01 
 1 (mild) 4 (80%) 5 (45%)    
 2 (moderate) 1 (20%) 4 (36%) 3 (60%)   
 3 (severe)  1 (9%) 2 (40%)   
 4 (cirrhosis)      
Portal fibrosis      
 0 (none)  2 (18%)  ns P<0.01 
 1 (mild) 5 (100%) 7 (64%)    
 2 (moderate)   4 (80%)   
 3 (severe)  2 (18%) 1 (20%)   
 4 (cirrhosis)      
Steatosis (n=5)NASH (n=11)ASH (n=5)NASH compared with ControlASH compared with Control
Steatosis      
 0 (none)  1 (9%) 1 (20%) P<0.01 ns 
 1 (mild) 3 (60%) 3 (27%) 2 (40%)   
 2 (moderate) 1 (20%) 3 (27%) 1 (20%)   
 3 (severe) 1 (20%) 4 (36%) 1 (20%)   
Lobular inflammation      
 0 (none) 5 (100%)   P<0.01 P<0.05 
 1 (<2)*  1 (9%) 2 (40%)   
 2 (2–4)*  8 (73%) 2 (40%)   
 3 (>4)*  2 (18%) 1 (20%)   
Portal inflammation      
 0 (none)  2 (18%)  P<0.05 P<0.01 
 1 (mild) 5 (100%) 9 (82%) 4 (80%)   
 2 (moderate)      
 3 (severe)   1 (20%)   
Lobular fibrosis      
 0 (none)  1 (9%)  P<0.05 P<0.01 
 1 (mild) 4 (80%) 5 (45%)    
 2 (moderate) 1 (20%) 4 (36%) 3 (60%)   
 3 (severe)  1 (9%) 2 (40%)   
 4 (cirrhosis)      
Portal fibrosis      
 0 (none)  2 (18%)  ns P<0.01 
 1 (mild) 5 (100%) 7 (64%)    
 2 (moderate)   4 (80%)   
 3 (severe)  2 (18%) 1 (20%)   
 4 (cirrhosis)      

NAFLD patients display increased biological markers of necroptosis

Among chronic liver diseases, NAFLD is becoming increasingly recognized as an important public health concern with an important economic burden. Although pathogenesis remains incompletely understood, hepatocyte cell death has been shown to play a key role in disease progression [20]. We next further assessed the contribution of necroptosis during NAFLD pathogenesis. Markers of hepatocellular injury, namely transaminases and γ-GT, were elevated in the serum of patients with steatosis and, more significantly, in patients with NASH (at least, P<0.05; Figure 2A). Moreover, absolute ALT levels were higher than AST levels in NAFLD patients. To further explore the role of hepatic necrosis in NAFLD pathogenesis, we determined serum levels of diverse biomarkers of necrosis/necroptosis, previously shown to associate with liver damage [30,31]. Determination of circulating levels of cytokeratin 18 cleaved by caspases has been suggested as a blood biomarker for predicting the presence of NASH [30]. Similarly, CK18-M65, the major intermediate filament in the liver, passively released from necrotic cells into the extracellular compartment, were found significantly elevated in steatosis (P<0.05) and NASH (P<0.01), compared with healthy volunteers (Figure 2B). HMGB1 protein is a ubiquitous nuclear protein that is released by necrotic cells into the extracellular space, thus triggering an inflammatory response by interacting with different cellular receptors, like the toll-like receptors (TLRs). In fact, NAFLD patients displayed increased protein systemic levels of HMGB1 (P<0.01) and CypA (P<0.05), a proposed early marker of necroptosis [32]. In addition, TNF-α is a well-established modulator of necroptosis and contributes not only to the liver inflammatory process, but also to hepatic insulin resistance and fat accumulation associated with NASH [33,34]. Accordingly, serum levels of TNF-α were significantly increased in NASH patients, compared with steatosis (P<0.01) and controls (P<0.05; Figure 2C). Finally, MLKL phosphorylation by RIP3 kinase is specifically required for ensuring necroptotic cell death. We found that increased expression of RIP3 in the liver of NASH patients (P<0.01) is accompanied by an increase of MLKL phosphorylation (P<0.05; Figure 2D). Taken together, the enhanced liver levels of RIP3, accompanied by an increase of MLKL phosphorylation and serum markers of hepatic necrosis and TNF-α in NASH patients strongly suggest a role for necroptosis in NAFLD progression.

Human NAFLD patients display increased biological markers of hepatocyte injury and necroptosis

Figure 2
Human NAFLD patients display increased biological markers of hepatocyte injury and necroptosis

Hepatocyte injury and necroptosis markers in serum and liver of normal controls (n=5), patients with simple steatosis (n=29) and NASH (n=15). (A) Serum markers of hepatocellular injury ALT, AST and γGT. (B) Serum markers of necrosis CK18-M65, HMGB1 and CypA. (C) Serum levels of TNF-α. (D) Immunoblotting and densitometry of RIP3, p-MLKL and total MLKL in liver. Representative immunoblots of normal controls (1), patients with simple steatosis (2) and NASH (3) are shown. β-Actin was used as a loading control. Data are expressed as mean ± S.E.M. §P<0.05 and *P<0.01 from Control; P<0.01 compared with steatosis.

Figure 2
Human NAFLD patients display increased biological markers of hepatocyte injury and necroptosis

Hepatocyte injury and necroptosis markers in serum and liver of normal controls (n=5), patients with simple steatosis (n=29) and NASH (n=15). (A) Serum markers of hepatocellular injury ALT, AST and γGT. (B) Serum markers of necrosis CK18-M65, HMGB1 and CypA. (C) Serum levels of TNF-α. (D) Immunoblotting and densitometry of RIP3, p-MLKL and total MLKL in liver. Representative immunoblots of normal controls (1), patients with simple steatosis (2) and NASH (3) are shown. β-Actin was used as a loading control. Data are expressed as mean ± S.E.M. §P<0.05 and *P<0.01 from Control; P<0.01 compared with steatosis.

Liver necroptosis is activated in HFCD diet-induced experimental NASH

To further explore whether necroptosis is actively involved in liver damage during NAFLD, we assessed the role of liver necroptosis in an experimental mouse model of NASH. Adult C57BL/6 mice were fed a standard diet (control) or a HFCD diet for 6 and 18 weeks. This dietary model does not induce significant changes in growth-rate and body weight of the mice (result not shown). No macroscopic abnormalities were detected in the liver of pair-fed control animals. Conversely, after 6 and 18 weeks, livers from HFCD-fed mice showed typical features of steatohepatitis. Histological analysis of liver sections from mice fed a HFCD diet for 6 weeks revealed severe steatosis (P<0.01) and inflammation (P<0.01), without relevant fibrosis in mice fed a HFCD diet for 6 weeks, compared with controls. After 18 weeks, steatosis and inflammation were still severe, but also associated with hepatocellular ballooning and sinusoidal fibrosis (Figure 3A; Table 3), closely mimicking the histopathological features of human NASH. In fact, HFCD-fed mice also exhibited physiological changes related to disease progression, namely increased mRNA levels of proinflammatory cytokines TNF-α and IL-1β in the liver (P<0.05; Figure 3B).

Necroptosis is activated in the liver of a mouse model of steatohepatitis

Figure 3
Necroptosis is activated in the liver of a mouse model of steatohepatitis

Adult C57BL/6 mice were placed on standard (control) or HFCD diet for 6 and 18 weeks. (A) H&E and CAB staining of representative liver sections. Scale bar=50 μm. (B) qRT-PCR analysis of TNF-α and IL-1β in mouse liver. (C) Immunoblotting and densitometry of total RIP3 and MLKL. (D) RIP3 and MLKL in insoluble and soluble fractions of liver whole cell lysates. Blots were normalized to endogenous Ponceau S staining. Representative immunoblots are shown. Results are expressed as mean ± S.E.M. fold change of five individual mice. §P<0.05 and *P<0.01 compared with respective Control; P<0.05 compared with HFCD diet-fed mice for 6 weeks.

Figure 3
Necroptosis is activated in the liver of a mouse model of steatohepatitis

Adult C57BL/6 mice were placed on standard (control) or HFCD diet for 6 and 18 weeks. (A) H&E and CAB staining of representative liver sections. Scale bar=50 μm. (B) qRT-PCR analysis of TNF-α and IL-1β in mouse liver. (C) Immunoblotting and densitometry of total RIP3 and MLKL. (D) RIP3 and MLKL in insoluble and soluble fractions of liver whole cell lysates. Blots were normalized to endogenous Ponceau S staining. Representative immunoblots are shown. Results are expressed as mean ± S.E.M. fold change of five individual mice. §P<0.05 and *P<0.01 compared with respective Control; P<0.05 compared with HFCD diet-fed mice for 6 weeks.

Table 3
Histological data of the HFCD animal model

Histological features are presented as number and percentage of cases in each grade (%). Portal/septal fibrosis was graded 0 in all animal groups. Kruskal–Wallis non-parametric ANOVA was performed to assess differences between animal groups.

ControlHFCDHFCDHFCDHFCD
6/18 week (n=5/5)6 week (n=5)18 week (n=4)compared with control 6 weekcompared with control 18 week
Steatosis      
 0 (none) 5 (100%)     
 1 (mild)    P<0.01 ns 
 2 (moderate)   2 (50%)   
 3 (severe)  5 (100%) 2 (50%)   
Lobular inflammation      
 0 (none)      
 1 (mild) 5 (100%)   P<0.01 P<0.05 
 2 (moderate)   1 (25%)   
 3 (severe)  5 (100%) 3 (75%)   
Hepatocellular ballooning     
 0 (none) 5 (100%)     
 1 (mild)      
 2 (moderate)  5 (100%)  ns P<0.01 
 3 (severe)   4 (100%)   
Pericellular fibrosis      
 0 (none) 5 (100%)     
 1 (mild)  2 (40%)  ns P<0.01 
 2 (moderate)  3 (60%)    
 3 (severe)   4 (100%)   
ControlHFCDHFCDHFCDHFCD
6/18 week (n=5/5)6 week (n=5)18 week (n=4)compared with control 6 weekcompared with control 18 week
Steatosis      
 0 (none) 5 (100%)     
 1 (mild)    P<0.01 ns 
 2 (moderate)   2 (50%)   
 3 (severe)  5 (100%) 2 (50%)   
Lobular inflammation      
 0 (none)      
 1 (mild) 5 (100%)   P<0.01 P<0.05 
 2 (moderate)   1 (25%)   
 3 (severe)  5 (100%) 3 (75%)   
Hepatocellular ballooning     
 0 (none) 5 (100%)     
 1 (mild)      
 2 (moderate)  5 (100%)  ns P<0.01 
 3 (severe)   4 (100%)   
Pericellular fibrosis      
 0 (none) 5 (100%)     
 1 (mild)  2 (40%)  ns P<0.01 
 2 (moderate)  3 (60%)    
 3 (severe)   4 (100%)   

RIP3-dependent signalling was very recently suggested to play a role in hepatic cell death and fibrosis in an animal model of NASH [35]. However, RIP3 also mediates non-necroptotic pathways that could be involved in liver damage, including inflammasome activation and inflammatory cytokine activation [36]. In turn, MLKL is a specific and critical RIP3-dowstream effector of necroptosis. Our results showed that RIP3 and MLKL expression was strongly increased in whole liver cell lysates from mice fed HFCD diet for 18 weeks (P<0.05; Figure 3C), being preferentially sequestered in the insoluble protein fraction (Figure 3D). Although RIP3 and MLKL levels were similar between control and HFCD-diet groups at 6 weeks (Figure 3C), these proteins appeared to be more significantly retained in the insoluble protein fraction of the HFCD-fed mice livers, compared with the soluble fraction (Figure 3D). These findings are consistent with the known insoluble amyloid structure of the necrosome, critical in the transmission of the pro-necroptotic signal [2,37]. Overall, our results suggest that HFCD diet-induced liver damage involves activation of necroptosis at both 6 and 18 weeks of feeding. Still, RIP3- and MLKL-dependent signalling is more significant at 18 weeks, concomitantly with aggravated liver injury, suggesting that necroptosis is involved in NAFLD progression.

Palmitic acid and TNF-α induce necroptosis in primary rat hepatocytes

The activation of necroptosis in human and mouse NAFLD prompted us to further explore the mechanisms governing these actions in vitro, determining whether distinct relevant stimuli are able to trigger necroptosis. PA is a saturated non-esterified fatty acid contributing to NASH pathogenesis [38] and widely used in in vitro models of NASH [39]. In addition, TNF-α also plays a key role in NAFLD pathogenesis. Because activation of death receptors in cells with defective apoptotic machinery can lead to necroptosis [3], primary rat hepatocytes were loaded with either PA or TNF-α plus CHX, in the presence or absence of zVAD-fmk, to inhibit caspase activation. In parallel, cells were incubated with or without Nec-1, to further confirm whether necroptosis is a significant cell-death pathway in hepatocytes and, simultaneously, whether it can be effectively targeted, hinting at prospective novel therapeutic strategies for NAFLD and related inflammation-associated liver diseases. Both PA (Figure 4A) and TNF-α/CHX alone (Figure 4B) induced apoptosis of primary rat hepatocytes, as revealed by activation of the executioner caspases-3/-7 (P<0.05) and overall cell death (P<0.01; Figures 4A and 4B). Co-incubation of either PA or TNF-α/CHX with Nec-1 had no effect on caspase 3-dependent cell death. Interestingly, suppression of caspase-3/-7 activities using zVAD-fmk was not able to prevent overall cell death, despite completely inhibiting caspases activity (P<0.01; Figures 4A and 4B). In agreement, TNF-α/CHX alone induced apoptosis, whereas co-incubation with zVAD-fmk completely abrogated it (result not shown). These results suggest that, in conditions were apoptosis is blocked, hepatocytes can effectively shift between different cell death pathways to still eliminate injured cells. In the present study, because Nec-1 was effective at inhibiting cell death induced by co-incubation of either PA or TNF-α/CHX with zVAD-fmk (at least, P<0.05), the alternate cell-death pathway appears to be necroptosis. To confirm this, RIP3 expression was evaluated by immunocytochemistry in TNF-α/CHX-induced apoptotic and necroptotic hepatocytes (Figure 4C). Not surprisingly, RIP3 expression was not affected by Nec-1, as it mainly targets RIP1 kinase activity. Curiously, TNF-α/CHX induced a slight increase in RIP3 expression levels (P<0.05), compared with control cells. Still, this increase more than doubled in the presence of zVAD-fmk (P<0.05), consistent with the role of RIP3 in necroptosis. Further, in unstimulated or TNF-α/CHX-treated hepatocytes, RIP3 displayed a diffused pattern or occasionally accumulated in the nucleus (Figure 4C, upper panel). On the other hand and in agreement with the amyloid structure of the necrosome, a large number of cells displayed a RIP3 punctuated pattern within the cytoplasm, after TNF-α/CHX plus zVAD-fmk stimulation. Collectively, our results indicate that primary rat hepatocytes are vulnerable to necroptosis induced by insults involved in NAFLD pathogenesis, which in turn can be rescued by Nec-1.

PA and TNF-α induce necroptosis of primary rat hepatocytes

Figure 4
PA and TNF-α induce necroptosis of primary rat hepatocytes

Primary rat hepatocytes were incubated with PA (400 μM) or TNF-α (10 ng/ml)/CHX (0.5 μg/ml), in the presence or absence of zVAD-fmk (50 μM) and/or Nec-1 (100 μM) or vehicle control for 24 h. Cell death and caspase-3/-7 activity were assessed by the LDH assay and using the Caspase-Glo 3/7 Assay respectively. (A) PA-induced cell death and caspase-3/-7 activity. (B) TNF-α/CHX-induced cell death and caspase-3/7 activity. Results are expressed as mean ± S.E.M. fold change from at least three independent experiments. (C) Representative RIP3 immunostaining (red) and Hoechst 33258 staining (blue) of cells treated with TNF-α/CHX (upper panel). Quantification of RIP3 mean fluorescence intensity of primary rat hepatocytes treated with TNF-α/CHX (lower panel). Fluorescence intensity was normalized with the number of cells per microscopic field. Scale bar=50 μm. Results are expressed as mean ± S.E.M. fold change from eight random microscopic fields per sample of three independent experiments. §P<0.05 and *P<0.01 compared with Control; P<0.05 and P<0.01 compared with respective Control.

Figure 4
PA and TNF-α induce necroptosis of primary rat hepatocytes

Primary rat hepatocytes were incubated with PA (400 μM) or TNF-α (10 ng/ml)/CHX (0.5 μg/ml), in the presence or absence of zVAD-fmk (50 μM) and/or Nec-1 (100 μM) or vehicle control for 24 h. Cell death and caspase-3/-7 activity were assessed by the LDH assay and using the Caspase-Glo 3/7 Assay respectively. (A) PA-induced cell death and caspase-3/-7 activity. (B) TNF-α/CHX-induced cell death and caspase-3/7 activity. Results are expressed as mean ± S.E.M. fold change from at least three independent experiments. (C) Representative RIP3 immunostaining (red) and Hoechst 33258 staining (blue) of cells treated with TNF-α/CHX (upper panel). Quantification of RIP3 mean fluorescence intensity of primary rat hepatocytes treated with TNF-α/CHX (lower panel). Fluorescence intensity was normalized with the number of cells per microscopic field. Scale bar=50 μm. Results are expressed as mean ± S.E.M. fold change from eight random microscopic fields per sample of three independent experiments. §P<0.05 and *P<0.01 compared with Control; P<0.05 and P<0.01 compared with respective Control.

TNF-α induced necroptosis involves RIP3-dependent ROS production

To evaluate the functional role of RIP3 during primary rat hepatocyte necroptosis, cells were transfected with a siRNA specific for RIP3. RIP3 siRNA-transfected cells displayed a 60% decrease in total RIP3 protein levels, compared with siRNA-control transfected cells (P<0.01; Figure 5A). After RIP3 knockdown, cell death induced by TNF-α/CHX plus zVAD-fmk was abrogated (P<0.01), whereas no effects were observed in cells incubated with TNF-α/CHX alone (Figure 5B). These results confirm the specific role of RIP3 in hepatocyte necroptosis.

TNF-α-induced necroptosis of primary rat hepatocytes involves RIP3-dependent ROS production

Figure 5
TNF-α-induced necroptosis of primary rat hepatocytes involves RIP3-dependent ROS production

Primary rat hepatocytes were transfected with a siRNA targeting RIP3 (siRIP3) or control (siControl) for 48 h. Primary rat hepatocytes or primary mouse hepatocytes isolated from WT and RIP3−/− mice were pre-treated for 1 h with Nec-1 (100 μM) and/or zVAD-fmk (50 μM) or DMSO vehicle control before treatment with TNF-α (10 ng/ml)/CHX (0.5 μg/ml) for 3 h (ROS measurement) or 24 h (cell death assays). (A) Representative immunoblot of RIP3 silencing in primary rat hepatocytes. β-actin was used as a loading control. (B) Cell death in primary rat hepatocytes as assessed by LDH activity assay. Results are expressed as mean ± S.E.M. fold change from four independent experiments. *P<0.01 compared with Control and P<0.01 compared with respective Control. (C) Fluorescence intensity from primary rat hepatocytes stained with the fluorescent probe H2DCFDA was measured and the values corrected with total protein content. Results are expressed as mean ± S.E.M. fold change from four independent experiments. §P<0.05 from TNF-α/CHX; P<0.05 compared with TNF-α/CHX plus zVAD-fmk. All conditions of exposure of primary rat hepatocytes to TNF-α/CHX are at least P<0.05 compared with control. (D) Cell death in WT and RIP3−/− mouse hepatocytes as assessed by the LDH activity assay. (E) Fluorescence intensity from WT and RIP3−/− mouse hepatocytes stained with the fluorescent probe H2DCFDA was measured and the values corrected with total protein content. Results are expressed as mean ± S.E.M. fold change from three independent experiments. §P<0.05 compared with respective control.

Figure 5
TNF-α-induced necroptosis of primary rat hepatocytes involves RIP3-dependent ROS production

Primary rat hepatocytes were transfected with a siRNA targeting RIP3 (siRIP3) or control (siControl) for 48 h. Primary rat hepatocytes or primary mouse hepatocytes isolated from WT and RIP3−/− mice were pre-treated for 1 h with Nec-1 (100 μM) and/or zVAD-fmk (50 μM) or DMSO vehicle control before treatment with TNF-α (10 ng/ml)/CHX (0.5 μg/ml) for 3 h (ROS measurement) or 24 h (cell death assays). (A) Representative immunoblot of RIP3 silencing in primary rat hepatocytes. β-actin was used as a loading control. (B) Cell death in primary rat hepatocytes as assessed by LDH activity assay. Results are expressed as mean ± S.E.M. fold change from four independent experiments. *P<0.01 compared with Control and P<0.01 compared with respective Control. (C) Fluorescence intensity from primary rat hepatocytes stained with the fluorescent probe H2DCFDA was measured and the values corrected with total protein content. Results are expressed as mean ± S.E.M. fold change from four independent experiments. §P<0.05 from TNF-α/CHX; P<0.05 compared with TNF-α/CHX plus zVAD-fmk. All conditions of exposure of primary rat hepatocytes to TNF-α/CHX are at least P<0.05 compared with control. (D) Cell death in WT and RIP3−/− mouse hepatocytes as assessed by the LDH activity assay. (E) Fluorescence intensity from WT and RIP3−/− mouse hepatocytes stained with the fluorescent probe H2DCFDA was measured and the values corrected with total protein content. Results are expressed as mean ± S.E.M. fold change from three independent experiments. §P<0.05 compared with respective control.

Oxidative stress is a well-established mediator of liver injury in NAFLD pathogenesis [26,40]. Moreover, in specific cellular contexts, oxidative stress has also been suggested to play a crucial role in executing necroptosis [4,9]. A significant increase in ROS production was detected in primary rat hepatocytes exposed to TNF-α/CHX plus zVAD-fmk for 3 h, when compared with TNF-α/CHX alone and no addition (P<0.05; Figure 5C). Consistent with its effects on necrosome assembly, Nec-1 pre-treatment significantly counteracted this increase (P<0.05).

To further confirm the association between necroptosis, RIP3 and oxidative stress, cultured hepatocytes isolated from WT and RIP3−/− mice were used. Both TNF-α/CHX and TNF-α/CHX plus zVAD-fmk increased overall cell death and ROS levels in WT hepatocytes by ∼50% (Figures 5C and 5D). Of note, cell death and oxidative stress were completely abrogated upon incubation of RIP3−/− mouse hepatocytes with TNF-α/CHX plus zVAD-fmk but not TNF-α/CHX alone, confirming the specific critical role of RIP3 in necroptotic signalling. Altogether, oxidative stress is a downstream event of RIP3 activation and appears to play a mechanistic role during necroptotic signalling induced by TNF-α in hepatocytes.

Absence of RIP3 ameliorates hepatic damage during MCD diet-induced experimental NASH

To further evaluate the functionality of RIP3-dependent necroptosis in NASH, C57BL/6 WT and RIP3−/− mice were fed a MCD diet for either 2 or 8 weeks. This represents a traditional dietary mouse model of NASH that induces steatosis, inflammation and hepatic fibrosis. Serum ALT and AST levels were significantly increased in MCD diet-fed WT mice compared with control mice on chow diet, at both time-points (at least, P<0.05; Figure 6A). Importantly, circulating levels of hepatic enzymes were significantly reduced in RIP3−/− animals (P<0.05), indicating a role for RIP3 during MCD diet-induced hepatic injury. Next, we scored liver lesions and accumulation of lipid droplets in the hepatocytes, after MCD-diet feeding. WT and RIP3−/− mice on chow diet displayed normal liver morphology and minimal hepatocellular lipid accumulation. WT and RIP3−/− mice on the MCD diet for 2 weeks showed mild to moderate vacuolation of hepatocytes, corresponding to accumulation of small to medium-size lipid droplets, as seen with the Oil Red O staining. These lesions were of similar severity in both groups. In turn, WT mice on the MCD diet for 8 weeks showed moderate to severe vacuolation of hepatocytes, corresponding to accumulation of large lipid droplets, whereas RIP3−/− mice displayed significantly decreased hepatocyte vacuolation and smaller and scattered lipid droplets (P<0.01; Figures 6B and 6C).

RIP3 deficiency ameliorates liver injury, steatosis, inflammation and fibrosis in MCD diet-induced experimental NASH

Figure 6
RIP3 deficiency ameliorates liver injury, steatosis, inflammation and fibrosis in MCD diet-induced experimental NASH

C57BL/6 WT and RIP3−/− were allowed free access to MCD diet or pair-fed control diet (Control) for either 2 or 8 weeks. (A) Serum AST and ALT levels. (B) Representative images of H&E and Oil Red O-stained liver sections. Scale bar=100 μm. (C) Steatosis score in blinded liver samples. Steatosis was graded on a scale 0–3. (D) qRT-PCR analysis of TNF-α and IL-1β in mouse liver. (E) qRT-PCR analysis of TGFβ and collagen-1α1 in mouse liver. Results are expressed as mean ± S.E.M. fold change of five individual mice. §P<0.05 and *P<0.01 compared with Control diet-fed mice; P<0.05 and P<0.01 compared with MCD diet-fed WT mice at respective time-point. (F) Representative images of Masson's Trichrome stained liver sections from MCD or control diet-fed mice for 8 weeks. Scale bar=100 μm.

Figure 6
RIP3 deficiency ameliorates liver injury, steatosis, inflammation and fibrosis in MCD diet-induced experimental NASH

C57BL/6 WT and RIP3−/− were allowed free access to MCD diet or pair-fed control diet (Control) for either 2 or 8 weeks. (A) Serum AST and ALT levels. (B) Representative images of H&E and Oil Red O-stained liver sections. Scale bar=100 μm. (C) Steatosis score in blinded liver samples. Steatosis was graded on a scale 0–3. (D) qRT-PCR analysis of TNF-α and IL-1β in mouse liver. (E) qRT-PCR analysis of TGFβ and collagen-1α1 in mouse liver. Results are expressed as mean ± S.E.M. fold change of five individual mice. §P<0.05 and *P<0.01 compared with Control diet-fed mice; P<0.05 and P<0.01 compared with MCD diet-fed WT mice at respective time-point. (F) Representative images of Masson's Trichrome stained liver sections from MCD or control diet-fed mice for 8 weeks. Scale bar=100 μm.

Because inflammation is a key pathological feature of NASH, we assessed hepatic expression of proinflammatory mediators in both WT and RIP3−/− mice. MCD feeding induced a ∼2- and 3-fold increase in hepatic IL-1β and TNF-α mRNA levels respectively at 2 and 8 weeks, in WT mice. This was completely abrogated in the absence of RIP3 (at least, P<0.05; Figure 6D). Finally, as fibrosis is the most nefarious outcome of sustained liver damage and inflammation, we investigated whether absence of RIP3 was also reflected by a decrease in liver fibrosis. In fact, RIP3 deficiency strongly decreased hepatic mRNA levels of fibrosis marker colloagen-1α1 and pro-fibrogenic factor TGFβ induced by MCD feeding at both 2 and 8 weeks (at least, P<0.05; Figure 6E). In addition, Masson's Trichrome staining of liver sections of WT MCD-fed mice for 8 weeks revealed the presence of collagen deposition near the sinusoids and associated with mononuclear cell infiltration within liver parenchyma of mild to moderate severity. This contrasted with RIP3−/− mice on MCD diet and WT and RIP3−/− mice on chow diet, in which collagen staining was minimal and mostly restricted to portal areas (Figure 6F). Altogether, our results show that RIP3-dependent signalling promotes liver injury, hepatic steatosis, inflammation and fibrosis in murine NASH, thus contributing to NAFLD pathogenesis.

Activation of RIP3-dependent necroptosis and oxidative stress constitute early events in MCD-diet induced NASH

RIP3-dependent signalling can promote proinflammatory cytokine production independently of necroptosis activation [36]. The fact that RIP3 deficiency abrogates cytokine expression after 2 weeks of MCD feeding without significantly affecting lipid deposition prompted us to further confirm whether RIP3-dpendent necroptosis is, in fact, activated at early time-points of MCD diet-induced NASH. Because MLKL and RIP3 oligomerization is a pre-requisite for necroptosis execution, we evaluated total and soluble/insoluble levels of RIP3 and MLKL in liver cell lysates from MCD diet-fed mice for 2 weeks. The MCD diet increased RIP3 and MLKL expression in whole liver cell lysates from WT mice compared with controls (at least, P<0.05; Figure 7A). More importantly, RIP3 and MLKL were accumulated in the insoluble protein fraction of WT mouse livers after MCD feeding (P<0.05; Figures 7B and 7C). Remarkably, although the increase in total hepatic MLKL expression was similar between WT and RIP3−/− mice after MCD feeding (Figure 7A), absence of RIP3 prevented sequestration of MLKL in the insoluble protein fraction of the livers (Figures 7B and 7C). These findings suggest that RIP3 is critical to MLKL oligomerization in the liver in response to MCD diet and, hence, necroptosis is activated early in this NASH model.

MCD diet induces RIP3-dependent MLKL sequestration in insoluble liver protein fractions and oxidative stress at early time-points

Figure 7
MCD diet induces RIP3-dependent MLKL sequestration in insoluble liver protein fractions and oxidative stress at early time-points

C57BL/6 WT and RIP3−/− were allowed free access to MCD diet or pair-fed control diet (Control) for two weeks. (A) Immunoblotting and densitometry of total RIP3 and MLKL. (B) RIP3 and MLKL in insoluble fractions of liver whole cell lysates. (C) RIP3 and MLKL in soluble fractions of liver whole cell lysates. Representative immunoblots are shown. Blots were normalized to endogenous Ponceau S staining. (D) Fluorescence intensity from whole cell lysates stained with the fluorescent probe H2DCFDA was measured and the values corrected with total protein content. Results are expressed as mean ± S.E.M. fold change of five individual mice. §P<0.05 and *P<0.01 compared with Control diet-fed mice and P<0.01 compared with MCD diet-fed WT mice.

Figure 7
MCD diet induces RIP3-dependent MLKL sequestration in insoluble liver protein fractions and oxidative stress at early time-points

C57BL/6 WT and RIP3−/− were allowed free access to MCD diet or pair-fed control diet (Control) for two weeks. (A) Immunoblotting and densitometry of total RIP3 and MLKL. (B) RIP3 and MLKL in insoluble fractions of liver whole cell lysates. (C) RIP3 and MLKL in soluble fractions of liver whole cell lysates. Representative immunoblots are shown. Blots were normalized to endogenous Ponceau S staining. (D) Fluorescence intensity from whole cell lysates stained with the fluorescent probe H2DCFDA was measured and the values corrected with total protein content. Results are expressed as mean ± S.E.M. fold change of five individual mice. §P<0.05 and *P<0.01 compared with Control diet-fed mice and P<0.01 compared with MCD diet-fed WT mice.

Finally, since MCD diet-induced NASH is associated with increased oxidative stress and our mechanistic studies showed a link between RIP3-dependent necroptosis and ROS production, we further assessed whether RIP3 deficiency modulates the oxidative stress response in vivo. Our results showed that absence of RIP3 strongly decreased ROS generation in the liver after MCD for 2 weeks compared with WT mice (P<0.05; Figure 7D). These findings confirm that oxidative stress is a downstream event resulting from activation of necroptosis during NAFLD-associated liver injury.

DISCUSSION

The relevance of apoptosis in the pathogenesis of inflammation-driven liver diseases, such as NAFLD, has been extensively documented. Interestingly, necroptosis is a well-orchestrated form of programmed cell death, sharing upstream signalling elements of apoptosis and morphological characteristics of necrosis. Although necroptosis activation has recently been implicated in a variety of pathological conditions [3,1013], its importance in the pathogenesis of inflammatory liver diseases has been poorly explored. In the present study, we show that necroptosis is also a common and important cell death pathway in hepatocytes and a pathological feature of NAFLD, contributing to inflammation and liver damage.

RIP3 kinase represents a crucial mediator of necroptosis and its protein expression levels correlate with cell sensitivity to necroptosis [10,11,16]. Interestingly, we found that RIP3 is weakly expressed in hepatocytes. Thus, it is likely that necroptosis does not occur under physiological conditions in the liver. However, our results also show that RIP3 is significantly induced in human hepatocytes from patients with chronic liver disease and, as such, it may contribute to their enhanced susceptibility to necroptosis under these pathological conditions, with concomitant effects in liver injury. In fact, we show that RIP3 induction strongly correlates with steatohepatitis severity. In support, recent reports found elevated RIP3 expression levels in liver biopsies from patients with alcoholic liver disease [14] and NASH [35], suggesting links between RIP3-dependent necroptosis and animal liver injury. In addition, activation of RIP3-dependent necroptosis in animal models of drug-induced hepatotoxicity and acute liver failure has also been reported [14,16]. Still, our results are the first to show that necroptosis is a common pathological feature in a range of human inflammation-associated liver diseases. Of note, because RIP3 may also execute necroptosis-independent functions during inflammation [36], we cannot yet fully exclude the possibility that other RIP3-associated events are simultaneously activated in chronic liver diseases, contributing to disease pathogenesis.

Given the increasingly worldwide prominence of NAFLD, its association with inflammation and the poor knowledge of the pathogenic mechanisms governing its progression from simple steatosis to advanced NASH and, further, to cirrhosis and end-stage liver failure, we focused our analysis on the pathogenic role of hepatocyte necroptosis using human samples and experimental murine models of NAFLD. Our results showed that RIP3 expression was significantly increased in liver tissue from patients with NASH, compared with control individuals and only slightly increased in patients with steatosis. The mechanism by which RIP3 executes necrotic cell death involves MLKL phosphorylation and subsequent oligomerization and translocation to cellular membranes, causing its lysis. Thus, MLKL is a key player in necroptotic signalling and its increased phosphorylation is a specific marker of necroptosis activation. Intriguingly, MLKL phosphorylation in liver and serum markers of necrosis were increased in both steatosis and NASH patients. These results suggest that hepatocyte necroptosis is triggered in the absence of significantly increased levels of RIP3 expression and that increased RIP3 protein levels immediately follow. Further, they also hint at the possibility of using CypA as an early indicator of NAFLD in patients with metabolic syndrome. This was corroborated in two different dietary in vivo models of NAFLD. We first showed that the HFCD diet induced histopathological alterations in mouse liver that closely resembles those observed in human NASH, including severe steatosis, inflammation and fibrosis. In fact, similarly to the observations in the NAFLD human liver samples, mice on the HFCD diet displayed steatohepatitis after 6 weeks of feeding and, yet, RIP3 overall levels remained unchanged. However, RIP3 and MLKL were found significantly sequestered in the insoluble protein fraction of liver lysates, providing strong evidence of necrosome assembly and necroptosis activation [2,37]. At 18 weeks of feeding, steatohepatitis was aggravated and RIP3 and MLKL strongly expressed and retained in the insoluble protein fraction of mice liver lysates. Again, these results suggest that necroptosis is engaged prior to highly increased RIP3 protein expression levels, contributing to leakage of pro-inflammatory mediators, such as CyPA and HMGB1. Upon a sustained stimulus, in this case the HFCD diet, it is likely that the increasing TNF-α production exacerbates necroptosis-associated liver injury and hence aggravates liver inflammation. Indeed, we demonstrated that RIP3 deficiency abrogates MCD-induced TNF-α expression, whereas TNF-α may induce necroptosis of cultured primary murine hepatocytes. Moreover, absence of RIP3 also attenuates hepatic inflammation, steatosis, liver injury and fibrosis after MCD feeding. Finally, it has been reported that Nec-1 is able to rescue MCD diet-mediated liver injury in mice [41]. Altogether, these findings strongly indicate that necroptosis is a key pathogenic factor involved in NAFLD triggering and progression. Importantly, we showed for the first time that mice on the MCD diet for 2 weeks, when steatohepatitis is quickly and strongly developing, display increased RIP3 protein levels, accompanied by RIP3-dependent MLKL sequestration in insoluble liver protein fractions. These findings further suggest that the assessment of RIP3 and MLKL protein levels in soluble/insoluble proteins fractions might be considered a valuable approach to characterize and identify necroptosis activation in vivo, filling the gap of lack of available specific biomarkers of necroptosis and excluding the involvement of non-necroptotic RIP3 functions.

With evidences that necroptosis is an active process during NAFLD, we also sought to elucidate some of the mechanisms underlying its activation. Our results showed that TNF-α is increased in serum of obese NASH patients and in the liver of NASH animals, in agreement with previous reports [33,42]. Further, TNF-α levels correlate with liver disease severity [43] and tumour necrosis factor receptor 1 (TNFR1) is overexpressed in livers of patients with NASH [43,44], whereas TNFR1-deficient mice are resistant to steatosis and liver injury in animal models of stetohepatitis [42]. Therefore, given its key role in necroptosis, TNF-α may trigger necroptotic signalling pathways in hepatocytes, through stimulation of TNFR1, thus mediating NAFLD-related hepatocyte injury. In agreement with this hypothesis, we show that incubation of primary hepatocytes with TNF-α/CHX plus a pancaspase inhibitor triggered cellular necroptosis. Moreover, it also promoted RIP3 up-regulation, which was also observed in human pathological liver samples and in in vivo models of steatohepatitis. Likewise, our results also show that co-incubation of PA and a pancaspase inhibitor also induce necroptosis in primary rat hepatocytes. Alongside TNF-α and PA, other stimuli are probably involved in triggering necroptosis in the NAFLD context. For instance, during NASH, hepatocytes are heavily exposed to TLR4 ligands, including the intestine-derived lipopolysaccharide (LPS), non-esterified fatty acids and HMGB1 [45]. In turn, activation of TLR4 in hepatocytes may trigger necroptosis by activating RIP3 through toll IL-1 receptor (TIR) domain-containing adaptor-inducing interferon-β (TRIF) [46].

Of note, in vitro, necroptosis appears to be mainly activated only when apoptosis is suppressed in the presence of zVAD-fmk, as Nec-1 had little protective effect in cells incubated with TNF-α/CHX alone. The apparent lack of relevance of necroptosis in vitro might have masked the importance of this cell-death pathway regarding the pathogenesis of liver diseases. Nevertheless, despite our evidence highlighting the role of necroptosis during NAFLD progression, apoptosis remains a well-established key feature of NAFLD [20,21]. In fact, necroptosis and apoptosis may be simultaneously activated in a liver disease context [16], specifically in NAFLD. In agreement, both genetic and pharmacological inhibition of caspases, in mouse models of NASH, was shown to be unable to completely abrogate hepatocyte cell death and oxidative stress, as well as liver inflammation and hepatic steatosis [40,47].

RIP3 has been shown to be the main switch responsible for shifting TNF-α-induced cell death from apoptosis to necroptosis in NIH3T3 mouse fibroblasts, in part by increasing metabolism-associated ROS generation [4]. Accordingly, we found that exposure of primary murine hepatocytes to TNF-α/CHX plus zVAD-fmk also triggers a RIP3-dependent ROS burst. In fact, although the link between ROS production and necroptosis appears to be context- and cell-specific [3], it has been shown that necroptotic liver injury in response to acetaminophen involves mitochondrial dysfunction and oxidative stress [15]. In addition, ROS plays a pathogenic role in NAFLD and predictors of oxidative stress have been found in both patients [26] and animal models of NASH [40]. Interestingly, these markers of oxidative stress positively correlate with necroinflammation in NASH patients, independently of the steatosis grade [48]. Free radicals may originate from activated lipid metabolism during NAFLD pathogenesis. However, oxidative stress may have causes other than changes in lipid metabolism. In fact, our results also show that ROS generation is a downstream event of RIP3 activation after MCD feeding for 2 weeks. Altogether, these studies corroborate our findings that oxidative stress is critical for the necroptosis signalling pathway in hepatocytes and further highlight that this is a mechanism occurring in vivo, during NASH pathogenesis and progression.

Unlike a previous publication [35], we found that MCD-induced hepatic lipid accumulation is significantly attenuated by RIP3 deficiency at 8 weeks. In agreement with our findings, it has been reported that absence of RIP3 reduced ethanol-induced hepatic steatosis by unknown mechanisms [14]. A possible explanation is that absence of RIP3 prevents oxidative damage and cytokine up-regulation, altering hepatic fat metabolism [20], and hence attenuates steatosis. Despite the current NAFLD burden, no specific and effective pharmacological therapy is yet available. Steatosis is usually benign, but can progress to NASH, increasing the risk of liver-related morbidity and mortality. To develop novel therapeutic strategies for NAFLD treatment, a better understanding of the underlying molecular, cellular and biochemical mechanisms implicated in the onset and progression of the disease is imperative. The present study underscores the presence and active role of RIP3-dependent necroptosis during NAFLD triggering and progression, among other inflammation-driven liver diseases. Further, mechanistic studies established the association between TNF-α-induced RIP3 expression, activation of necroptosis and oxidative stress in hepatocytes, shedding new light on disease pathogenesis. The targeting of liver necroptotic signalling pathways could arise as a promising therapeutic option to arrest NAFLD and other inflammation-associated liver diseases' development and progression.

AUTHOR CONTRIBUTIONS

Cecília Rodrigues was the principal investigator of the study responsible for study concept and design. Marta Afonso was involved in performing the experiments, analysis and data interpretation and writing. Pedro Rodrigues and Marta Caridade helped in animal experiments. Tânia Carvalho and Paula Borralho were involved in animal histology analysis. Rui Castro was involved in the planning and organization of the study and critical revision of the manuscript. Helena Cortez-Pinto was responsible for collection and selection of human specimens.

The authors thank Dr Elisa Alves from Clinical Analyses Core Laboratory and Dr Manuela Gaspar from Animal Facilities, Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal for their support. We thank to Dr Vishva Dixit, Molecular Oncology Department, Genentech, Inc. (San Fransisco, CA, U.S.A.) and Dr Soares, Instituto Gulbenkian de Ciência (Oeiras, Portugal) for kindly providing RIP3−/− mice. Dr Soares and Dr Gozzelino, Instituto Gulbenkian de Ciência, and Dr Ferreira, Karolinska Institute (Stockholm, Sweden) are thanked for valuable help in optimizing primary mouse hepatocytes cultures. Finally, we thank all members of the laboratory for insightful discussions.

FUNDING

This work was supported by Fundação para a Ciência e a Tecnologia (grant numbers PTDC/SAU-ORG/119842/2010 and HMSP-ICT/0018/2011, and fellowship SFRH/BD/91119/2012) and Gilead Sciences [grant number PGG-028-2013].

Abbreviations

     
  • ALT

    alanine aminotransferase

  •  
  • ASH

    alcoholic steatohepatitis

  •  
  • AST

    aspartate aminotransferase

  •  
  • CAB

    chromotrope-aniline blue

  •  
  • CHX

    cycloheximide

  •  
  • CK-M65

    cytokeratin 18 full-length

  •  
  • CypA

    cyclophilin A

  •  
  • H&E

    haematoxylin and eosin

  •  
  • H2DCFDA

    2′,7′-dichlorodihydrofluorescein diacetate

  •  
  • HDL

    high-density lipoprotein

  •  
  • HFCD

    high-fat choline-deficient

  •  
  • HMGB1

    high-mobility group box 1

  •  
  • HPRT

    hypoxanthine phosphoribosyltransferase

  •  
  • IL

    interleukin

  •  
  • LDH

    lactate dehydrogenase

  •  
  • LDL

    low-density lipoprotein

  •  
  • MCD

    methionine and choline-deficient

  •  
  • MLKL

    mixed lineage kinase domain-like

  •  
  • NAFLD

    non-alcoholic fatty liver disease

  •  
  • NASH

    non-alcoholic steatohepatitis

  •  
  • Nec-1

    necrostatin-1

  •  
  • PA

    palmitic acid

  •  
  • p-MLKL

    phospho-MLKL

  •  
  • RIP

    receptor-interacting protein

  •  
  • RIPA

    radio-immunoprecipitation assay

  •  
  • ROS

    reactive oxygen species

  •  
  • TGF

    transforming growth factor

  •  
  • TLR

    toll-like receptor

  •  
  • TNFR1

    tumour necrosis factor receptor1

  •  
  • TNF-α

    tumour necrosis factor-α

  •  
  • WT

    wild-type

  •  
  • γ-GT

    γ-glutamyl transpeptidase

References

References
1
Guicciardi
 
M.E.
Malhi
 
H.
Mott
 
J.L.
Gores
 
G.J.
 
Apoptosis and necrosis in the liver
Compr. Physiol.
2013
, vol. 
3
 (pg. 
977
-
1010
)
[PubMed]
2
Li
 
J.
McQuade
 
T.
Siemer
 
A.B.
Napetschnig
 
J.
Moriwaki
 
K.
Hsiao
 
Y.S.
Damko
 
E.
Moquin
 
D.
Walz
 
T.
McDermott
 
A.
, et al 
The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis
Cell
2012
, vol. 
150
 (pg. 
339
-
350
)
[PubMed]
3
Degterev
 
A.
Huang
 
Z.
Boyce
 
M.
Li
 
Y.
Jagtap
 
P.
Mizushima
 
N.
Cuny
 
G.D.
Mitchison
 
T.J.
Moskowitz
 
M.A.
Yuan
 
J.
 
Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury
Nat. Chem. Biol.
2005
, vol. 
1
 (pg. 
112
-
119
)
[PubMed]
4
Zhang
 
D.W.
Shao
 
J.
Lin
 
J.
Zhang
 
N.
Lu
 
B.J.
Lin
 
S.C.
Dong
 
M.Q.
Han
 
J.
 
RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis
Science
2009
, vol. 
325
 (pg. 
332
-
336
)
[PubMed]
5
Upton
 
J.W.
Kaiser
 
W.J.
Mocarski
 
E.S.
 
Virus inhibition of RIP3-dependent necrosis
Cell Host Microbe
2010
, vol. 
7
 (pg. 
302
-
313
)
[PubMed]
6
Moujalled
 
D.M.
Cook
 
W.D.
Okamoto
 
T.
Murphy
 
J.
Lawlor
 
K.E.
Vince
 
J.E.
Vaux
 
D.L.
 
TNF can activate RIPK3 and cause programmed necrosis in the absence of RIPK1
Cell Death Dis.
2013
, vol. 
4
 pg. 
e465
 
[PubMed]
7
McQuade
 
T.
Cho
 
Y.
Chan
 
F.K.
 
Positive and negative phosphorylation regulates RIP1- and RIP3-induced programmed necrosis
Biochem. J.
2013
, vol. 
456
 (pg. 
409
-
415
)
[PubMed]
8
Wang
 
H.
Sun
 
L.
Su
 
L.
Rizo
 
J.
Liu
 
L.
Wang
 
L.F.
Wang
 
F.S.
Wang
 
X.
 
Mixed lineage kinase domain-like protein MLKL causes necrotic membrane disruption upon phosphorylation by RIP3
Mol. Cell
2014
, vol. 
54
 (pg. 
133
-
146
)
[PubMed]
9
Zhao
 
J.
Jitkaew
 
S.
Cai
 
Z.
Choksi
 
S.
Li
 
Q.
Luo
 
J.
Liu
 
Z.G.
 
Mixed lineage kinase domain-like is a key receptor interacting protein 3 downstream component of TNF-induced necrosis
Proc. Natl. Acad. Sci. U.S.A.
2012
, vol. 
109
 (pg. 
5322
-
5327
)
[PubMed]
10
He
 
S.
Wang
 
L.
Miao
 
L.
Wang
 
T.
Du
 
F.
Zhao
 
L.
Wang
 
X.
 
Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha
Cell
2009
, vol. 
137
 (pg. 
1100
-
1111
)
[PubMed]
11
Gunther
 
C.
Martini
 
E.
Wittkopf
 
N.
Amann
 
K.
Weigmann
 
B.
Neumann
 
H.
Waldner
 
M.J.
Hedrick
 
S.M.
Tenzer
 
S.
Neurath
 
M.F.
Becker
 
C.
 
Caspase-8 regulates TNF-alpha-induced epithelial necroptosis and terminal ileitis
Nature
2011
, vol. 
477
 (pg. 
335
-
339
)
[PubMed]
12
Welz
 
P.S.
Wullaert
 
A.
Vlantis
 
K.
Kondylis
 
V.
Fernandez-Majada
 
V.
Ermolaeva
 
M.
Kirsch
 
P.
Sterner-Kock
 
A.
van Loo
 
G.
Pasparakis
 
M.
 
FADD prevents RIP3-mediated epithelial cell necrosis and chronic intestinal inflammation
Nature
2011
, vol. 
477
 (pg. 
330
-
334
)
[PubMed]
13
Bonnet
 
M.C.
Preukschat
 
D.
Welz
 
P.S.
van Loo
 
G.
Ermolaeva
 
M.A.
Bloch
 
W.
Haase
 
I.
Pasparakis
 
M.
 
The adaptor protein FADD protects epidermal keratinocytes from necroptosis in vivo and prevents skin inflammation
Immunity
2011
, vol. 
35
 (pg. 
572
-
582
)
[PubMed]
14
Roychowdhury
 
S.
McMullen
 
M.R.
Pisano
 
S.G.
Liu
 
X.
Nagy
 
L.E.
 
Absence of receptor interacting protein kinase 3 prevents ethanol-induced liver injury
Hepatology
2013
, vol. 
57
 (pg. 
1773
-
1783
)
[PubMed]
15
Ramachandran
 
A.
McGill
 
M.R.
Xie
 
Y.
Ni
 
H.M.
Ding
 
W.X.
Jaeschke
 
H.
 
Receptor interacting protein kinase 3 is a critical early mediator of acetaminophen-induced hepatocyte necrosis in mice
Hepatology
2013
, vol. 
58
 (pg. 
2099
-
2108
)
[PubMed]
16
Vucur
 
M.
Reisinger
 
F.
Gautheron
 
J.
Janssen
 
J.
Roderburg
 
C.
Cardenas
 
D.V.
Kreggenwinkel
 
K.
Koppe
 
C.
Hammerich
 
L.
Hakem
 
R.
, et al 
RIP3 inhibits inflammatory hepatocarcinogenesis but promotes cholestasis by controlling caspase-8- and JNK-dependent compensatory cell proliferation
Cell Rep.
2013
, vol. 
4
 (pg. 
776
-
790
)
[PubMed]
17
Liedtke
 
C.
Bangen
 
J.M.
Freimuth
 
J.
Beraza
 
N.
Lambertz
 
D.
Cubero
 
F.J.
Hatting
 
M.
Karlmark
 
K.R.
Streetz
 
K.L.
Krombach
 
G.A.
, et al 
Loss of caspase-8 protects mice against inflammation-related hepatocarcinogenesis but induces non-apoptotic liver injury
Gastroenterology
2011
, vol. 
141
 (pg. 
2176
-
2187
)
[PubMed]
18
Zhou
 
Y.
Dai
 
W.
Lin
 
C.
Wang
 
F.
He
 
L.
Shen
 
M.
Chen
 
P.
Wang
 
C.
Lu
 
J.
Xu
 
L.
, et al 
Protective effects of necrostatin-1 against concanavalin A-induced acute hepatic injury in mice
Mediators Inflamm.
2013
, vol. 
2013
 pg. 
706156
 
[PubMed]
19
Malhi
 
H.
Guicciardi
 
M.E.
Gores
 
G.J.
 
Hepatocyte death: a clear and present danger
Physiol. Rev.
2010
, vol. 
90
 (pg. 
1165
-
1194
)
[PubMed]
20
Ferreira
 
D.M.
Simao
 
A.L.
Rodrigues
 
C.M.
Castro
 
R.E.
 
Revisiting the metabolic syndrome and paving the way for microRNAs in non-alcoholic fatty liver disease
FEBS J.
2014
, vol. 
281
 (pg. 
2503
-
2524
)
[PubMed]
21
Ferreira
 
D.M.
Castro
 
R.E.
Machado
 
M.V.
Evangelista
 
T.
Silvestre
 
A.
Costa
 
A.
Coutinho
 
J.
Carepa
 
F.
Cortez-Pinto
 
H.
Rodrigues
 
C.M.
 
Apoptosis and insulin resistance in liver and peripheral tissues of morbidly obese patients is associated with different stages of non-alcoholic fatty liver disease
Diabetologia
2011
, vol. 
54
 (pg. 
1788
-
1798
)
[PubMed]
22
Pinto
 
H.C.
Baptista
 
A.
Camilo
 
M.E.
Valente
 
A.
Saragoca
 
A.
de Moura
 
M.C.
 
Nonalcoholic steatohepatitis. Clinicopathological comparison with alcoholic hepatitis in ambulatory and hospitalized patients
Dig. Dis. Sci.
1996
, vol. 
41
 (pg. 
172
-
179
)
[PubMed]
23
Mariash
 
C.N.
Seelig
 
S.
Schwartz
 
H.L.
Oppenheimer
 
J.H.
 
Rapid synergistic interaction between thyroid hormone and carbohydrate on mRNAS14 induction
J. Biol. Chem.
1986
, vol. 
261
 (pg. 
9583
-
9586
)
[PubMed]
24
Castro
 
R.E.
Ferreira
 
D.M.
Zhang
 
X.
Borralho
 
P.M.
Sarver
 
A.L.
Zeng
 
Y.
Steer
 
C.J.
Kren
 
B.T.
Rodrigues
 
C.M.
 
Identification of microRNAs during rat liver regeneration after partial hepatectomy and modulation by ursodeoxycholic acid
Am. J. Physiol. Gastrointest. Liver Physiol.
2010
, vol. 
299
 (pg. 
G887
-
G897
)
[PubMed]
25
Richieri
 
G.V.
Kleinfeld
 
A.M.
 
Unbound free fatty acid levels in human serum
J. Lipid Res.
1995
, vol. 
36
 (pg. 
229
-
240
)
[PubMed]
26
Sanyal
 
A.J.
Campbell-Sargent
 
C.
Mirshahi
 
F.
Rizzo
 
W.B.
Contos
 
M.J.
Sterling
 
R.K.
Luketic
 
V.A.
Shiffman
 
M.L.
Clore
 
J.N.
 
Nonalcoholic steatohepatitis: association of insulin resistance and mitochondrial abnormalities
Gastroenterology
2001
, vol. 
120
 (pg. 
1183
-
1192
)
[PubMed]
27
Belfort
 
R.
Harrison
 
S.A.
Brown
 
K.
Darland
 
C.
Finch
 
J.
Hardies
 
J.
Balas
 
B.
Gastaldelli
 
A.
Tio
 
F.
Pulcini
 
J.
, et al 
A placebo-controlled trial of pioglitazone in subjects with nonalcoholic steatohepatitis
N. Engl. J. Med.
2006
, vol. 
355
 (pg. 
2297
-
2307
)
[PubMed]
28
Simoes
 
A.E.
Pereira
 
D.M.
Amaral
 
J.D.
Nunes
 
A.F.
Gomes
 
S.E.
Rodrigues
 
P.M.
Lo
 
A.C.
D'Hooge
 
R.
Steer
 
C.J.
Thibodeau
 
S.N.
, et al 
Efficient recovery of proteins from multiple source samples after TRIzol((R)) or TRIzol((R))LS RNA extraction and long-term storage
BMC Genomics
2013
, vol. 
14
 pg. 
181
 
[PubMed]
29
Castro
 
R.E.
Ferreira
 
D.M.
Afonso
 
M.B.
Borralho
 
P.M.
Machado
 
M.V.
Cortez-Pinto
 
H.
Rodrigues
 
C.M.
 
miR-34a/SIRT1/p53 is suppressed by ursodeoxycholic acid in the rat liver and activated by disease severity in human non-alcoholic fatty liver disease
J. Hepatol.
2013
, vol. 
58
 (pg. 
119
-
125
)
[PubMed]
30
Eguchi
 
A.
Wree
 
A.
Feldstein
 
A.E.
 
Biomarkers of liver cell death
J. Hepatol.
2014
, vol. 
60
 (pg. 
1063
-
1074
)
[PubMed]
31
Dear
 
J.W.
Simpson
 
K.J.
Nicolai
 
M.P.
Catterson
 
J.H.
Street
 
J.
Huizinga
 
T.
Craig
 
D.G.
Dhaliwal
 
K.
Webb
 
S.
Bateman
 
D.N.
Webb
 
D.J.
 
Cyclophilin A is a damage-associated molecular pattern molecule that mediates acetaminophen-induced liver injury
J. Immunol.
2011
, vol. 
187
 (pg. 
3347
-
3352
)
[PubMed]
32
Christofferson
 
D.E.
Yuan
 
J.
 
Cyclophilin A release as a biomarker of necrotic cell death
Cell Death Differ.
2010
, vol. 
17
 (pg. 
1942
-
1943
)
[PubMed]
33
Li
 
Z.
Yang
 
S.
Lin
 
H.
Huang
 
J.
Watkins
 
P.A.
Moser
 
A.B.
Desimone
 
C.
Song
 
X.Y.
Diehl
 
A.M.
 
Probiotics and antibodies to TNF inhibit inflammatory activity and improve nonalcoholic fatty liver disease
Hepatology
2003
, vol. 
37
 (pg. 
343
-
350
)
[PubMed]
34
Valenti
 
L.
Fracanzani
 
A.L.
Dongiovanni
 
P.
Santorelli
 
G.
Branchi
 
A.
Taioli
 
E.
Fiorelli
 
G.
Fargion
 
S.
 
Tumor necrosis factor alpha promoter polymorphisms and insulin resistance in nonalcoholic fatty liver disease
Gastroenterology
2002
, vol. 
122
 (pg. 
274
-
280
)
[PubMed]
35
Gautheron
 
J.
Vucur
 
M.
Reisinger
 
F.
Vargas Cardenas
 
D.
Roderburg
 
C.
Koppe
 
C.
Kreggenwinkel
 
K.
Schneider
 
A.T.
Bartneck
 
M.
Neumann
 
U.P.
, et al 
A positive feedback loop between RIP3 and JNK controls non-alcoholic steatohepatitis
EMBO Mol. Med.
2014
, vol. 
6
 (pg. 
1062
-
1074
)
[PubMed]
36
Vince
 
J.E.
Wong
 
W.W.
Gentle
 
I.
Lawlor
 
K.E.
Allam
 
R.
O'Reilly
 
L.
Mason
 
K.
Gross
 
O.
Ma
 
S.
Guarda
 
G.
, et al 
Inhibitor of apoptosis proteins limit RIP3 kinase-dependent interleukin-1 activation
Immunity
2012
, vol. 
36
 (pg. 
215
-
227
)
[PubMed]
37
Wang
 
Z.
Jiang
 
H.
Chen
 
S.
Du
 
F.
Wang
 
X.
 
The mitochondrial phosphatase PGAM5 functions at the convergence point of multiple necrotic death pathways
Cell
2012
, vol. 
148
 (pg. 
228
-
243
)
[PubMed]
38
Araya
 
J.
Rodrigo
 
R.
Videla
 
L.A.
Thielemann
 
L.
Orellana
 
M.
Pettinelli
 
P.
Poniachik
 
J.
 
Increase in long-chain polyunsaturated fatty acid n - 6/n - 3 ratio in relation to hepatic steatosis in patients with non-alcoholic fatty liver disease
Clin. Sci.
2004
, vol. 
106
 (pg. 
635
-
643
)
[PubMed]
39
Cazanave
 
S.C.
Mott
 
J.L.
Bronk
 
S.F.
Werneburg
 
N.W.
Fingas
 
C.D.
Meng
 
X.W.
Finnberg
 
N.
El-Deiry
 
W.S.
Kaufmann
 
S.H.
Gores
 
G.J.
 
Death receptor 5 signaling promotes hepatocyte lipoapoptosis
J. Biol. Chem.
2011
, vol. 
286
 (pg. 
39336
-
39348
)
[PubMed]
40
Hatting
 
M.
Zhao
 
G.
Schumacher
 
F.
Sellge
 
G.
Al Masaoudi
 
M.
Gabetaler
 
N.
Boekschoten
 
M.
Muller
 
M.
Liedtke
 
C.
Cubero
 
F.J.
Trautwein
 
C.
 
Hepatocyte caspase-8 is an essential modulator of steatohepatitis in rodents
Hepatology
2013
, vol. 
57
 (pg. 
2189
-
2201
)
[PubMed]
41
von Montfort
 
C.
Matias
 
N.
Fernandez
 
A.
Fucho
 
R.
Conde de la Rosa
 
L.
Martinez-Chantar
 
M.L.
Mato
 
J.M.
Machida
 
K.
Tsukamoto
 
H.
Murphy
 
M.P.
, et al 
Mitochondrial GSH determines the toxic or therapeutic potential of superoxide scavenging in steatohepatitis
J. Hepatol.
2012
, vol. 
57
 (pg. 
852
-
859
)
[PubMed]
42
Tomita
 
K.
Tamiya
 
G.
Ando
 
S.
Ohsumi
 
K.
Chiyo
 
T.
Mizutani
 
A.
Kitamura
 
N.
Toda
 
K.
Kaneko
 
T.
Horie
 
Y.
, et al 
Tumour necrosis factor alpha signalling through activation of Kupffer cells plays an essential role in liver fibrosis of non-alcoholic steatohepatitis in mice
Gut
2006
, vol. 
55
 (pg. 
415
-
424
)
[PubMed]
43
Crespo
 
J.
Cayon
 
A.
Fernandez-Gil
 
P.
Hernandez-Guerra
 
M.
Mayorga
 
M.
Dominguez-Diez
 
A.
Fernandez-Escalante
 
J.C.
Pons-Romero
 
F.
 
Gene expression of tumor necrosis factor alpha and TNF-receptors, p55 and p75, in nonalcoholic steatohepatitis patients
Hepatology
2001
, vol. 
34
 (pg. 
1158
-
1163
)
[PubMed]
44
Ribeiro
 
P.S.
Cortez-Pinto
 
H.
Sola
 
S.
Castro
 
R.E.
Ramalho
 
R.M.
Baptista
 
A.
Moura
 
M.C.
Camilo
 
M.E.
Rodrigues
 
C.M.
 
Hepatocyte apoptosis, expression of death receptors, and activation of NF-kappaB in the liver of nonalcoholic and alcoholic steatohepatitis patients
Am. J. Gastroenterol.
2004
, vol. 
99
 (pg. 
1708
-
1717
)
[PubMed]
45
Roh
 
Y.S.
Seki
 
E.
 
Toll-like receptors in alcoholic liver disease, non-alcoholic steatohepatitis and carcinogenesis
J. Gastroenterol. Hepatol.
2013
, vol. 
28
 
Suppl 1
(pg. 
38
-
42
)
[PubMed]
46
He
 
S.
Liang
 
Y.
Shao
 
F.
Wang
 
X.
 
Toll-like receptors activate programmed necrosis in macrophages through a receptor-interacting kinase-3-mediated pathway
Proc. Natl. Acad. Sci. U.S.A.
2011
, vol. 
108
 (pg. 
20054
-
20059
)
[PubMed]
47
Anstee
 
Q.M.
Concas
 
D.
Kudo
 
H.
Levene
 
A.
Pollard
 
J.
Charlton
 
P.
Thomas
 
H.C.
Thursz
 
M.R.
Goldin
 
R.D.
 
Impact of pan-caspase inhibition in animal models of established steatosis and non-alcoholic steatohepatitis
J. Hepatol.
2010
, vol. 
53
 (pg. 
542
-
550
)
[PubMed]
48
Seki
 
S.
Kitada
 
T.
Sakaguchi
 
H.
 
Clinicopathological significance of oxidative cellular damage in non-alcoholic fatty liver diseases
Hepatol. Res.
2005
, vol. 
33
 (pg. 
132
-
134
)
[PubMed]