Interleukin-32 (IL-32) is a cytokine and it showed a protective role in liver damage caused by chronic alcohol abuse through reducing oxidative stress and inflammatory responses.

CLINICAL PERSPECTIVES

  • IL-32 is a cytokine closely associated with various inflammatory disease; however, no study reported that the role of IL-32 in ethanol-induced liver disease. IL-32γ is the most active isoform among IL-32 isofoms.

  • In our study, IL-32γ mice showed attenuated ethanol-induced liver injury through reducing oxidative stress and inflammatory responses.

  • These results suggest that IL-32γ may play a protective role in alcoholic liver damage by chronic alcohol abuse.

INTRODUCTION

Alcoholic liver disease (ALD) caused by excessive and chronic alcohol consumption is a chronic liver disease and approximately 100 000 deaths are related to alcohol abuse each year in the United States of America [1]. In 1960s, Lieber et al. [2] showed that alcohol is a hepatotoxin and that ALD is not caused by malnutrition by showing that alcohol produces a fatty liver despite an adequate diet. ALD presents complex disease states, ranging from the benign form of fatty liver to severe forms of alcoholic hepatitis, cirrhosis and hepatocellular carcinoma. The earliest fatty liver stage develops in approximately 90%–100% of long-term heavy drinkers [3], but only about 30% of heavy drinkers develop more severe forms such as fibrosis, cirrhosis and hepatocellular carcinoma [4].

Ethanol is primarily metabolized by the alcohol dehydrogenase and cytochrome P450 2E1 (CYP2E1) pathways. CYP2E1 is the key enzyme of the hepatic microsomal ethanol-oxidizing system, but persistent alcohol abuse leads to liver injury caused by toxic products of alcohol metabolism such as acetaldehyde and 1-hydroxyethyl radicals [5]. CYP2E1 is an effective generator of reactive oxygen species (ROS), because its catalytic activity requires oxygen activation [6]. ROS are important signal transduction molecules involved in metabolic pathways; however, a high concentration of ROS is toxic to the liver because of DNA damage, mitochondrial dysfunction and lipid peroxidation caused by ROS reactions [7]. Lu et al. [8] reported that CYP2E1 contributes to ethanol-induced fatty liver in mice and ethanol-induced liver injury is attenuated in CYP2E1-knockout mice. Chronic ethanol exposure also increases blood nitric oxide (NO) by inducing inducible nitric oxide synthase (iNOS) expression; this contributes to ethanol-induced liver injury and mitochondrial dysfunction in the liver [911].

Nuclear transcription factor κB (NF-κB) is a key regulator of various genes involved in inflammation and immune cell infiltration [12]. Chronic alcohol consumption not only leads to a fatty liver but also induces innate immunity through the translocation of bacteria-derived lipopolysaccharide (LPS) from the gut to the liver [13]. LPS in the liver binds to Toll-like receptor 4 and affects NF-κB activity through an intracellular signalling cascade [14]. Furthermore, oxidative stress activates NF-κB. ROS activate NF-κB by release of inhibitor of NF-κB (IκB) and antioxidants inhibit NF-κB activation in many cell types [15]. Thus, alcohol abuse leads to the up-regulation of inflammatory cytokines in the liver by NF-κB activation, resulting in liver injury.

Peroxisome-proliferator-activated receptors (PPARs) consist of three subtypes (PPARα, PPARγ and PPARδ/β); they are transcription factors involved in lipid homoeostasis [16]. PPARα is primarily expressed in the brown adipose tissue, liver, kidney, heart and skeletal muscle and regulates genes involved in fatty acid transcription and oxidation [17]. PPARβ/δ is found in various tissues and promotes adipocyte differentiation by induction of PPARγ [18]. PPARγ is mainly expressed in adipose tissue and is involved in adipocyte differentiation and lipid storage [19]. According to recently published studies, alcohol consumption is associated with the function of PPARs [20,21]. Activation of PPARγ by rosiglitazone, a PPARγ agonist, attenuated ethanol-induced hepatosteatosis [20] and liver failure by alcohol consumption [21]. Thus, alcohol abuse leads to the dysregulation of lipid metabolism in the liver. PPARs are also associated with NF-κB activation. In T-cells, PPARγ activators inhibit inflammatory cytokine expression via inhibition of NF-κB [22] and PPARα increases IκB expression [23].

The NK4 transcript (natural killer cell transcript 4), renamed interleukin (IL)-32, was first identified as a highly up-regulated NK gene activated by IL-2 or mitogen-activated T-cells [24], but numerous studies have since reported that IL-32 is expressed in various cell types such as endothelial, epithelial, macrophage and dendritic cells [25]. It was initially determined to induce tumour necrosis factor (TNF)-α [26], but is also associated with infectious disease, chronic inflammation and cancer [25]. Six isoforms of IL-32 have been described, namely IL-32α, IL-32β, IL-32γ, IL-32δ, IL-32ε and IL-32ζ [26,27]. Although IL-32β expression seems to be most abundant [27], IL-32γ is the most active form [28]. Humans and other mammals, such as pigs, cows and horses, express the IL32 gene, but IL-32 has still not been found in rodents such as mice and rats [25]. Many efforts indicate that the IL32 gene is located between MMP25 and ZSCAN10 gene in rodents; however, a large part of the IL32 gene is missing and its functional transcription is still unknown [25]. Furthermore, although several studies about the function of IL-32 have been reported, IL-32 signalling remains unclear. Many studies including our studies have reported the effect of IL-32 using IL-32 transgenic mice [29,30]. We previously reported that IL-32γ inhibits acetaminophen-induced hepatotoxicity through NF-κB inactivation [31]. Therefore, we expected that IL-32γ attenuates ethanol-induced hepatotoxicity too. In the present study, we investigated the effects of IL-32γ on chronic ethanol-induced liver injury in IL-32γ transgenic mice.

MATERIALS AND METHODS

Transgenesis and animal experiments

We previously generated IL-32γ-overexpressing transgenic mice (IL-32γ mice), and identified that IL-32γ was ubiquitously expressed in various tissues such as liver, kidney, intestine, spleen, brain, heart and thymus [30]. Briefly, to generate IL-32β transgenic mice, the human IL-32γ cDNA was subcloned into the mammalian expression vector pCAGGS and this construct was used for IL-32γ transgenic mice production. Reverse transcription (RT)-PCR and Western blotting revealed that IL-32γ was ubiquitously expressed in various tissues in IL-32γ mice but there was no expression of IL-32γ in the tissues of wild-type (WT) mice. Age-matched male mice with IL-32γ and C57BL/6 background (10–12 weeks old) were used in all of the experiments. Mice were randomized into four dietary groups (n=6). Each group of mice received two different types of liquid diets for 6 weeks: (1) paired-fed standard diet with water (control diet); (2) alcohol diet with ethanol (ethanol diet). Ethanol comprised 35.8% of the total calories in mice receiving ethanol. Liquid diets (control diet and ethanol diet) were based upon the Lieber–DeCarli ethanol formulation and were purchased from DYETS Inc. The ethanol concentration was kept thereafter at 6.6% for 6 weeks. After 6 weeks of feeding, mice were killed. All studies were approved by and performed according to the ethical guidelines by the Chungbuk National University Animal Care Committee (CBNU-523-13-01).

Measurements of serum aspartate transaminase and alanine transaminase

Mice were anaesthetized with an overdose of pentobarbital (100 mg/kg) and blood was taken by heart puncture. Serum levels of aspartate transaminase (AST) and alanine transaminase (ALT) were measured at the Laboratory Animal Research Center of Chungbuk National University.

Histological techniques

For histological processing, liver tissues were fixed in phosphate buffer containing 10% formaldehyde and decalcified with EDTA. Fixed tissues were processed by routine methods to paraffin blocks. Specimens were sectioned at 4 μm and stained with haematoxylin and eosin stain (H&E).

Western blot analysis

Homogenized liver tissues and human hepatic cells lysed by protein extraction solution (PRO-PREP, iNtRON Biotechnology) containing protease inhibitor cocktail (Calbiochem) and phosphatase inhibitor cocktail (Roche). Total proteins (30 μg) were separated by SDS/PAGE and transferred to a PVDF membrane (Millipore). The membrane was blocked with 5% dried skimmed milk overnight and then incubated with primary antibodies (diluted 1:1000) for 1 h at room temperature. The membranes were immunoblotted with the following primary antibodies: mouse monoclonal antibodies directed against PPARγ, PPARα, iNOS, CYP2E1, intercellular adhesion molecule 1 (ICAM-1) and β-actin (Santa Cruz Biotechnology) and against AMPK (5′ adenosine monophosphate-activated protein kinase), phospho-AMPK and cyclo-oxygenase-2 (COX-2) (Cell Signaling Technology). After washing with Tris-buffered saline containing 0.05% Tween-20 (TBST), the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies (diluted 1:3000) for 1 h at room temperature. Binding of antibodies to the PVDF membrane was detected with enhanced chemiluminescence solution (GE Healthcare) and X-ray film (Agfa).

RNA isolation and quantitative real-time RT-PCR

Total RNA was isolate from gastrocnemius muscle using TRIzol (Invitrogen). Samples were reverse-transcribed using ProSTAR™ (Stratagene). Gene expression analysis was performed by RT-PCR using QuantiNova SYBR Green PCR kit (Qiagen).

Nitro oxide and hydrogen peroxide assay

Nitro oxides were measured according to the manufacturer's instructions (iNtRON). Hydrogen peroxides were measured according to the manufacturer's instructions (Cell Biolabs). To perform assay, the liver tissue and human hepatic cells were homogenized, then normalized to protein concentration.

Reagents and cell culture

The HepG2 and Huh7 human hepatic cells were obtained from the American Type Culture Collection. HepG2 and Huh7 cells were grown at 37°C in 5% CO2-humidified air in Dulbecco's modified Eagle's medium (DMEM) that contained 10% FBS, 100 units/ml penicillin and 100 mg/ml streptomycin. DMEM, penicillin, streptomycin and FBS were purchased from Gibco Life Technologies. To isolate primary hepatocytes from mice liver tissue, WT and IL-32γ mice at the same time were killed by halothane inhalation. The mice were doused with 70% ethanol to minimize contamination of the primary cultures. Livers were removed from both mice using scissors and forceps soaked in 70% ethanol and as each organ was removed it was immediately placed in a 100-mm tissue culture dish containing 10 ml of sterile PBS. Livers were minced separately into 1-mm cubes using razor blades dipped in 70% ethanol. The minced tissues were transferred into sterile 15-ml conical tubes containing sterile PBS. After allowing the minced tissue pieces to settle, the PBS was aspirated and the tissues washed once more with sterile PBS. DMEM (20 ml) containing collagenase (2.5 mg/ml, Sigma) and DNase I (50 μl; Invitrogen) was added and the cells were incubated with rocking at 37°C for 3 h. After the incubation, collagenase-digested tissue and dissociated cells from tissue were centrifuged at 800 g for 5 min then the supernant was rejected. The resulting pellet was gently pressed through a 100 μm cell strainer (Becton, Dickinson & Company). The filtered cells were washed with DMEM and plated into 100 mm2 dishes. To develop an ethanol-induced oxidative stress model, primary hepatocytes, HepG2 and Huh7 cells were incubated with ethanol (100 mM) or without for 24 h.

Transfection

Human IL-32γ-6xmyc sequences were PCR-amplified from cDNA and subcloned into the EcoRI and XhoI sites of pcDNA3.1+. Transiently IL-32γ-overexpressing HepG2 or Huh7 cells were constructed by transfection with pcDNA3.1+-6xmyc or pcDNA3.1+IL-32β-6xmyc using Lipofectamine 2000 (Invitrogen).

Immunohistochemistry

All specimens were fixed in formalin and embedded in paraffin for examination. Sections (4 μm thickness) were stained with H&E and analysed by immunohistochemistry using primary mouse monoclonal antibodies directed against cluster of differentiation (CD) 8a, CD57 and iNOS (1:100 dilution), primary rabbit polyclonal antibody directed against COX-2 and CYP2E1 (1:100), primary rat monoclonal antibody directed against F4/80 (1:100) and secondary biotinylated anti-mouse, anti-rabbit and anti-rat antibodies.

Gel EMSA

DNA-binding activity of NF-κB was determined by gel EMSA. Gel EMSA was performed according to the manufacturer's recommendation (Promega). The liver tissues and cells were briefly homogenized in 200 μl of solution A (10 mM HEPES, pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol and 0.2 mM phenylmethylsulfonylfluoride), incubated on ice for 6 min and then centrifuged at 4000 g for 6 min. Pelleted nuclei were re-suspended in solution C (solution A supplemented with 420 mM NaCl and 20% glycerol) and incubated on ice with vigorous vortexing every 5 min for 20 min. The resuspended pellet was centrifuged at 18900 g for 15 min and the resulting nuclear extract supernatants were collected in a chilled micro tube. Consensus oligonucleotides were end-labelled using T4 polynucleotide kinase and [32P]ATP for 10 min at 37°C. Gel shift reaction mixtures were assembled and incubated at room temperature. Subsequently, 1 μl of gel loading buffer was added to each reaction and loaded on to a 6% non-denaturing gel. The gel was subjected to electrophoresis until the dye was four-fifths of the way down the gel. The gel was dried for 2 h at 80°C and exposed to film overnight at −70°C.

Cytokine assay

Liver tissues homogenized with protein extraction solution (PRO-PREP, iNtRON Biotechnology) and measured quantity of IL-6 in total proteins (1 mg) using mouse cytokine assay kit (R&D Systems).

RESULTS

IL-32γ transgenic mice ameliorated ethanol-induced hepatosteatosis and liver damage

Chronic ethanol exposure induces hepatosteatosis and liver damage [32]. To explore the effects of IL-32γ overexpression on liver damage in ethanol-fed mice, WT and IL-32γ mice were fed on a standard diet with water (control diet) or ethanol (ethanol diet). We observed that IL-32γ was not expressed in the liver of WT mice but IL-32γ mice and the expression of IL-32γ did not change by ethanol diet (Figure 1A). Histopathology studies revealed significant hepatosteatosis in ethanol-diet WT mice, but its manifestations were attenuated in ethanol-diet IL-32γ mice (Figure 1B). We observed large fat droplets in the livers of ethanol-diet WT mice and the size of the fat droplets was smaller in the livers of ethanol-diet IL-32γ mice. In addition, serum AST levels in WT mice were higher in ethanol-diet WT mice than in IL-32γ mice, although serum ALT levels did not differ significantly between the two groups (Figure 1C).

Transgenic IL-32γ mice are protected from ethanol-induced fatty liver diseases

Figure 1
Transgenic IL-32γ mice are protected from ethanol-induced fatty liver diseases

(A) The expression of IL-32γ in the liver of WT and IL-32γ mice on control or ethanol (EtOH) diet for 6 weeks (scale bars, 100 μm). (B) Liver sections of WT mice and IL-32γ mice on control or ethanol diet for 6 weeks were stained with H&E (scale bars, 100 μm). (C) Serum AST and ALT levels in WT mice and IL-32γ mice on a control or ethanol diet for 6 weeks. n=8 per group; means ± S.E.M.; *P < 0.05, control diet compared with ethanol diet in WT mice, #P<0.05, WT mice compared with IL-32γ mice.

Figure 1
Transgenic IL-32γ mice are protected from ethanol-induced fatty liver diseases

(A) The expression of IL-32γ in the liver of WT and IL-32γ mice on control or ethanol (EtOH) diet for 6 weeks (scale bars, 100 μm). (B) Liver sections of WT mice and IL-32γ mice on control or ethanol diet for 6 weeks were stained with H&E (scale bars, 100 μm). (C) Serum AST and ALT levels in WT mice and IL-32γ mice on a control or ethanol diet for 6 weeks. n=8 per group; means ± S.E.M.; *P < 0.05, control diet compared with ethanol diet in WT mice, #P<0.05, WT mice compared with IL-32γ mice.

Chronic ethanol-induced CYP2E1 expression and oxidative stress were reduced in IL-32γ transgenic mice

As illustrated in Figure 2(A), immunohistochemistry showed an induction of CYP2E1 in WT mice on an ethanol diet. However, the livers of ethanol-fed IL-32γ mice displayed significantly weaker CYP2E1 staining compared with ethanol-diet WT mice and this result was confirmed by a Western blot analysis (Figures 2A and 2B). Because the activation of CYP2E1 increases the production of ROS, we evaluated hydrogen peroxide levels. The hydrogen peroxide levels in the livers of ethanol-diet WT mice were higher than in the control-diet WT mice; however, the increase in hydrogen peroxide levels caused by ethanol was inhibited in the livers of IL-32γ mice (Figure 2C). NO production and iNOS expression were higher in the livers of ethanol-diet WT mice than in control-diet mice, but were not higher in the livers of ethanol-diet IL-32γ mice (Figures 2B and 2C).

Ethanol-induced oxidative stress was reduced in the liver of IL-32γ mice on ethanol diet

Figure 2
Ethanol-induced oxidative stress was reduced in the liver of IL-32γ mice on ethanol diet

(A) Immunohistochemical analysis of CYP 2E1 and iNOS confirmed in the liver tissues of WT mice and IL-32γ mice on control or ethanol (EtOH) diet (scale bars, 100 μm). (B) The expression of CYP 2E1 and iNOS were determined in the total protein extracts of mice liver tissues by Western blotting. (C) NO and hydrogen peroxide levels measured in the liver of WT mice and IL-32γ mice on control or ethanol diet. n=8 per group; means ± S.E.M.; *P < 0.05, control diet compared with ethanol diet in WT mice, #P<0.05, WT mice compared with IL-32γ mice.

Figure 2
Ethanol-induced oxidative stress was reduced in the liver of IL-32γ mice on ethanol diet

(A) Immunohistochemical analysis of CYP 2E1 and iNOS confirmed in the liver tissues of WT mice and IL-32γ mice on control or ethanol (EtOH) diet (scale bars, 100 μm). (B) The expression of CYP 2E1 and iNOS were determined in the total protein extracts of mice liver tissues by Western blotting. (C) NO and hydrogen peroxide levels measured in the liver of WT mice and IL-32γ mice on control or ethanol diet. n=8 per group; means ± S.E.M.; *P < 0.05, control diet compared with ethanol diet in WT mice, #P<0.05, WT mice compared with IL-32γ mice.

Ethanol-induced CYP2E1 expression and oxidative stress were reduced in primary hepatocytes from IL-32γ mice and human hepatic cells overexpressing IL-32γ

Since ethanol-induced oxidative stress was reduced in the livers of IL-32γ mice, we examined the effect of IL-32γ on ethanol-induced oxidative stress in primary hepatocytes from mouse liver. Primary hepatocytes from WT mice showed a marked increase in hydrogen peroxide levels and CYP2E1 expression following ethanol treatment for 24 h; however, primary hepatocytes from IL-32γ mice showed significantly lower hydrogen peroxide levels and CYP2E1 expression compared with those observed in primary hepatocytes from WT mice (Figures 3A and 3B). To investigate the effect of IL-32γ overexpression on ethanol-induced oxidative stress in human hepatic cells, we transfected human hepatic HepG2 or Huh7 cells with an IL-32γ-expressing vector (Figures 3C–3F). As shown Figures 3(C) and 3(E), ethanol-induced hydrogen peroxide levels were inhibited in IL-32γ-overexpressing HepG2 and Huh7 cells. Furthermore, ethanol-induced CYP2E1 expression decreased in IL-32γ-overexpressing HepG2 and Huh7 cells (Figures 3D and 3F). However, NO production was undetectable in HepG2 and Huh7 cells with or without ethanol (results not shown).

Ethanol-induced oxidative stress was reduced in primary hepatocytes from WT mice and IL-32γ mice, human hepatic HepG2 and Huh 7 cells

Figure 3
Ethanol-induced oxidative stress was reduced in primary hepatocytes from WT mice and IL-32γ mice, human hepatic HepG2 and Huh 7 cells

Intracellular hydrogen peroxide levels in (A) primary hepatocytes from WT and IL-32γ mice and in control or IL-32γ-overexpressing (C) Huh7 and (E) HepG2 cells treated with ethanol (EtOH) or without. Means ± S.E.M.; *P < 0.05, treated with compared with treated without ethanol in control cells, #P<0.05, control compared with IL-32γ-over expressing cells treated with ethanol. (B) The expression of CYP2E1 in primary hepatocytes from WT and IL-32γ mice treated with ethanol or without. The expression of CYP2E1 or transfected IL-32γ in control or IL-32γ-overexpressing (D) Huh7 and (F) HepG2 cells treated with ethanol (EtOH) or without.

Figure 3
Ethanol-induced oxidative stress was reduced in primary hepatocytes from WT mice and IL-32γ mice, human hepatic HepG2 and Huh 7 cells

Intracellular hydrogen peroxide levels in (A) primary hepatocytes from WT and IL-32γ mice and in control or IL-32γ-overexpressing (C) Huh7 and (E) HepG2 cells treated with ethanol (EtOH) or without. Means ± S.E.M.; *P < 0.05, treated with compared with treated without ethanol in control cells, #P<0.05, control compared with IL-32γ-over expressing cells treated with ethanol. (B) The expression of CYP2E1 in primary hepatocytes from WT and IL-32γ mice treated with ethanol or without. The expression of CYP2E1 or transfected IL-32γ in control or IL-32γ-overexpressing (D) Huh7 and (F) HepG2 cells treated with ethanol (EtOH) or without.

Ethanol-induced hepatic inflammation was reduced in IL-32γ transgenic mice

Because chronic ethanol exposure induces liver inflammation and injury, we determined COX-2 expression and pro-inflammatory cytokine levels for the livers of WT mice and IL-32γ mice. COX-2-reactive cells and COX-2 expression were significantly higher in the livers of ethanol-diet WT mice than in those of IL-32γ mice (Figures 4A and 4B). The expression of PPARα was decreased in the livers of both ethanol-diet WT mice and IL-32γ mice and the two groups did not differ significantly (Figure 4B). However, the expression of PPARγ was higher in the livers of ethanol-diet IL-32γ mice than in those of ethanol-diet WT mice (Figure 4B). The level of IL-6 was significantly higher in the livers of ethanol-diet WT mice than in those of control-diet WT mice and ethanol-diet IL-32γ mice (Figure 4C). Furthermore, ethanol-induced mRNA expression levels of other inflammatory cytokines such as interferon (IFN)-γ and monocyte chemoattractant protein-1 (MCP-1) in the liver of IL-32γ mice were greatly lower than the liver of WT mice (Figure 4D). However, TNF-α, another major inflammatory cytokine, was detected at very low levels and did not differ significantly between groups (results not shown).

Ethanol-induced COX-2 and IL-6 expression were decreased in the liver of IL-32γ mice

Figure 4
Ethanol-induced COX-2 and IL-6 expression were decreased in the liver of IL-32γ mice

(A) Immunohistochemical analysis of COX-2 in the liver tissues of WT mice and IL-32γ mice on control or ethanol diet (scale bars, 100 μm). (B) Expression of COX-2 in the liver tissues of WT mice and IL-32γ mice on control or ethanol (EtOH) diet. (C) Cytokine assay of the IL-6 in the livers of WT and IL-32γ mice on control or ethanol diet. mRNA expression levels of pro-inflammatory cytokines such as (D) IFN-γ and (E) MCP-1 in the livers of WT mice and IL-32γ mice on control and ethanol diet. n=8 per group; means ± S.E.M.; *P < 0.05, control diet compared with ethanol diet in WT mice, #P<0.05, WT mice compared with IL-32γ mice.

Figure 4
Ethanol-induced COX-2 and IL-6 expression were decreased in the liver of IL-32γ mice

(A) Immunohistochemical analysis of COX-2 in the liver tissues of WT mice and IL-32γ mice on control or ethanol diet (scale bars, 100 μm). (B) Expression of COX-2 in the liver tissues of WT mice and IL-32γ mice on control or ethanol (EtOH) diet. (C) Cytokine assay of the IL-6 in the livers of WT and IL-32γ mice on control or ethanol diet. mRNA expression levels of pro-inflammatory cytokines such as (D) IFN-γ and (E) MCP-1 in the livers of WT mice and IL-32γ mice on control and ethanol diet. n=8 per group; means ± S.E.M.; *P < 0.05, control diet compared with ethanol diet in WT mice, #P<0.05, WT mice compared with IL-32γ mice.

NF-κB activity was decreased in the liver of IL-32γ transgenic mice

To examine whether reduced liver inflammation was related to the inactivation of NF-κB in the ethanol-diet IL-32γ mice, the DNA-binding activity of NF-κB was determined by EMSA in the liver tissue. The DNA-binding activity of NF-κB was higher in the livers of ethanol-diet WT mice than in control-diet WT mice (Figure 5A). In the livers of IL-32γ mice, however, the DNA-binding activity of NF-κB was significantly less than that it was in the livers of ethanol-diet WT mice (Figure 5A). This DNA-binding activity of NF-κB was confirmed by Western blot analysis of the nuclear fraction of the mouse livers. The translocation of p65 and p60 into the nucleus was inhibited in the liver of ethanol-diet IL-32γ mice compared with ethanol-diet WT mice (Figure 5B). In human hepatic cells treated with ethanol, the DNA-binding activity of NF-κB was significantly less in IL-32γ-overexpressing cells than control cells (Figures 5C and 5D).

IL-32γ mice showed inhibition of NF-κB activity in the liver on ethanol diet

Figure 5
IL-32γ mice showed inhibition of NF-κB activity in the liver on ethanol diet

(A) The DNA-binding activity of NF-κB in the nuclear fraction was determined by EMSA in the liver of WT mice and IL-32γ mice on control or ethanol (EtOH) diet. (B) The nuclear translocation of p65 and p50 was determined by Western blotting in the liver of WT mice and IL-32γ mice on control or ethanol (EtOH) diet. The DNA-binding activity of NF-κB in the nuclear fraction was determined by EMSA in (C) Huh7 cells and (D) HepG2 cells transfected with IL-32γ or without (Con) and treated with ethanol (EtOH) or without.

Figure 5
IL-32γ mice showed inhibition of NF-κB activity in the liver on ethanol diet

(A) The DNA-binding activity of NF-κB in the nuclear fraction was determined by EMSA in the liver of WT mice and IL-32γ mice on control or ethanol (EtOH) diet. (B) The nuclear translocation of p65 and p50 was determined by Western blotting in the liver of WT mice and IL-32γ mice on control or ethanol (EtOH) diet. The DNA-binding activity of NF-κB in the nuclear fraction was determined by EMSA in (C) Huh7 cells and (D) HepG2 cells transfected with IL-32γ or without (Con) and treated with ethanol (EtOH) or without.

Infiltration of ethanol-induced immune cells was inhibited in IL-32γ transgenic mice

To investigate whether the inhibition of ethanol-induced hepatotoxicity in WT and IL-32γ mice was related to immune cell infiltration, we analysed the distribution of immune cells in liver tissues. More CD3 (a T-cell marker), CD57 (a natural killer cell marker) and F4/80 (a macrophage marker) were observed in the livers of ethanol-diet WT mice than in those of ethanol-diet IL-32γ mice (Figure 6A). These infiltrated cells were observed around the liver portal veins and expressed CYP2E1, iNOS and COX-2. The expression of ICAM-1 which is the adhesion molecule involved in leucocyte infiltration was significantly increased in the livers of ethanol-diet WT mice (Figure 6B). Expression of ICAM-1 was higher in the livers of IL-32γ mice than in the livers of control-diet WT mice and was lower in the livers of ethanol-diet IL-32γ mice (Figure 6B).

Ethanol-induced immune cells infiltration into the liver was inhibited in IL-32γ mice

Figure 6
Ethanol-induced immune cells infiltration into the liver was inhibited in IL-32γ mice

(A) Immunohistochemistry of infiltrated immune cells such as cytotoxic T-cells (CD8), natural killer cells (CD57) and macrophages (F4/80) in the liver of WT mice and IL-32γ mice on control or ethanol diet (scale bars, 100 μm). (B) ICAM-1, a major adhesion molecule, was determined in the liver of WT and IL-32γ mice on control or ethanol (EtOH) diet.

Figure 6
Ethanol-induced immune cells infiltration into the liver was inhibited in IL-32γ mice

(A) Immunohistochemistry of infiltrated immune cells such as cytotoxic T-cells (CD8), natural killer cells (CD57) and macrophages (F4/80) in the liver of WT mice and IL-32γ mice on control or ethanol diet (scale bars, 100 μm). (B) ICAM-1, a major adhesion molecule, was determined in the liver of WT and IL-32γ mice on control or ethanol (EtOH) diet.

DISCUSSION

We previously generated IL-32γ transgenic mice and reported ubiquitous expression of IL-32γ in various tissues [30]. In contrast, IL-32γ is not expressed in the tissues of non-transgenic mice [30]. We also reported that IL-32γ inhibits acetaminophen-induced acute hepatotoxicity [31]. In the present study, we found that ethanol-induced oxidative stress, liver inflammation and hepatosteatosis were attenuated in IL-32γ mice.

Chronic alcohol consumption leads to a fatty liver and liver injury. Ethanol exposure significantly induced hepatic steatosis in the livers of WT mice but it was attenuated in IL-32γ mice. In the liver tissue of ethanol-diet WT mice, large lipid droplets were revealed by lipid accumulation. The level of ALT, a major marker of hepatic damage in the serum, did not differ significantly between ethanol-diet WT mice and IL-32γ mice. A significantly elevated level of AST, another major marker of hepatic damage in the serum, was observed for ethanol-diet WT mice but not for ethanol-diet IL-32γ mice. Fatty liver disease is closely associated with oxidative stress. Metabolism by CYP2E1 generates ROS and CYP2E1 metabolizes increased polyunsaturated fatty acids to generate ω-hydroxylated fatty acids, which results in the generation of ROS [33]. Furthermore, induced oxidative stress by superoxide generation and mitochondrial dysfunction leads to fat accumulation in HepG2 cells [34]. Therefore, we investigated ethanol-induced oxidative stress in IL-32γ mice. Chronic ethanol exposure-induced CYP2E1 and iNOS expression leads to oxidative stress by the generation of ROS and increase in NO production in the liver, resulting in liver injury [6,10,11]. In the livers of IL-32γ mice, ethanol-induced CYP2E1 and iNOS expression did not increase. Moreover, the production of NO did not increase in the livers of ethanol-diet IL-32γ mice. ROS include the free radicals superoxide, hydroxyl radicals, hydrogen peroxide and ROS [33]. Of these, hydrogen peroxide is produced by CYP2E1 during ethanol metabolism and dismutation of superoxide, one of the ROS generated, by superoxide dismutase (SOD). Because hydrogen peroxide is more stable compared with other ROS, it may play an important role in the intracellular signalling. In the present study, hydrogen peroxide levels were elevated in the livers of ethanol-diet mice, but not in the livers of ethanol-induced IL-32γ mice. In addition, intracellular hydrogen peroxide level and expression of CYP2E1 were decreased in isolated primary hepatocytes from IL-32γ mice liver compared with WT mice liver. In human hepatic cells, an elevated intracellular hydrogen peroxide level and expression of CYP2E1 that occurs in response to ethanol was also lower in the IL-32γ-overexpressing HepG2 and Huh7 cells than in control HepG2 and Huh7 cells. Therefore, these results suggest that IL-32γ attenuated ethanol-induced liver injury through the inhibition of hydrogen peroxide generation via CYP2E1 expression.

Ethanol-induced liver damage is involved in inflammatory responses. IL-6, a major pro-inflammatory cytokine, is elevated by ethanol consumption and is closely associated with ALD [35]. IFN-γ is induced early by ethanol consumption [36] and induces proteasome activity through iNOS activation in the liver [37]. MCP-1 is a potent mononuclear cell-specific chemotactic protein and its production was increased in acute alcoholic hepatitis [38]. In our study, the level of IL-6 was significantly reduced in the liver of IL-32γ mice. Furthermore, other inflammatory cytokines such as IFN-γ and MCP-1 were also down-regulated in the liver of IL-32γ mice of both control- and ethanol-diet groups. We previously reported that acetaminophen-induced pro-inflammatory cytokines were down-regulated in the liver of IL-32γ mice [31]. Therefore, these results suggest that IL-32γ attenuates ethanol-induced pro-inflammatory cytokine production. COX-2 is up-regulated in ALD [39]. COX-2 is induced by LPS, pro-inflammatory cytokines and oxidant stress [39] and promotes prostaglandins, which are major inflammatory mediators [40]. iNOS expression is increased by chronic ethanol consumption and is also induced by pro-inflammatory cytokines [9]. Up-regulated iNOS expression leads to increased NO production resulting in liver damage by mitochondrial respiration dysfunction [9]. In the present study, ethanol-induced COX-2 and iNOS expression were inhibited in the liver of IL-32γ mice. These results suggest that IL-32γ inhibits ethanol-induced COX-2 and iNOS expression by down-regulation of pro-inflammatory cytokines. PPARs are also involved in ethanol-induced liver damage and inflammatory responses. Activation of PPARγ and elevation of its expression levels by rosiglitazone, a PPARγ agonist, ameliorates ethanol-induced hepatosteatosis [20]. Moreover, PPARα and PPARγ inhibit inflammatory responses by down-regulation of pro-inflammatory cytokines in monocytes [23] and NF-κB inactivation in skeletal muscle [41]. In our study, the expression of PPARγ, but not PPARα, increased in the livers of ethanol-diet IL-32γ mice compared with ethanol-diet WT mice. Thus, IL-32γ might partially inhibit inflammatory responses through disturbance of down-regulation of PPARγ by ethanol. Taken together, our results suggest that IL-32γ attenuates ethanol-induced liver injury through reduction in inflammatory responses.

NF-κB activation induces an inflammatory response by the regulation of inflammatory genes such as various cytokines, chemokines, cell adhesion molecules and immunoreceptors [42]. Chen et al. [43] reported that inhibitors of IκB-α phosphorylation selectively inhibit cytokine- and LPS-induced E-selectin, vascular cell adhesion protein-1 and ICAM-1 expression in human endothelial cells. Thus, NF-κB activation might play an important role in immune cell infiltration associated with the expression of cell adhesion molecules [43]. In the present study, ethanol-induced expression of ICAM-1 was decreased and the infiltration of immune cells such as T-cells, natural killer cells and macrophages was inhibited in the livers of IL-32γ mice. Thus, these results suggest that IL-32γ ameliorated ALDs by an inflammatory response through NF-κB inactivation. We previously reported that IL-32γ inhibits cancer growth and acetaminophen-induced liver injury by inactivation of NF-κB signals [30,31]. In the present study, IL-32γ mice and IL-32γ-over expressing human hepatic cells showed inactivated ethanol-induced NF-κB activity. These results suggest that IL-32γ may affect inactivation of ethanol-induced NF-κB. Although it remains to be investigated, it is worth noting that NF-κB is activated or inactivated by many cytokines. It has been reported that IL-6 induces the activation of NF-κB through phosphoinositide 3-kinase (PI3K)–Akt pathway in the intestinal epithelia, resulting in the induction of ICAM-1 expression [44]. TNF-α and IL-1β also induces the activation of NF-κB through NF-κB-inducible kinase (NIK) [45]. In contrast, IL-10 blocks NF-κB activity through blocked translocation of p65, which is one of the NF-κB subunits [46]. We found that the levels of IL-6 were significantly reduced and translocations of p65 as well as p50 were inhibited in ethanol-induced liver of IL-32γ mice. The acetylation and/or recruitment of p300 protein are critical in cytokine-induced activation of NF-κB and IL-6 prolongs their retention in the nucleus [47]. We previously reported that acetylation of p65 and expression of p300 in the nucleus of colon cancer cells were inhibited by the introduction of IL-32γ [30]. Thus, direct inhibition of acetylation and/or recruitment of p300 protein may partially contribute to the inhibitory effects of IL-32γ on the inactivation of NF-κB. Indirect inactivation via modulation of IL-6 may also play a role in the inhibitory effects of IL-32γ on the inactivation of NF-κB.

In summary, our results suggest that IL-32γ protects against ethanol-induced liver failure. This effect may result from a reduction in oxidative stress by inhibition of CYP2E1 expression and a reduction in inflammatory responses by the inactivation of NF-κB.

Abbreviations

     
  • ALD

    alcoholic liver disease

  •  
  • ALT

    alanine transaminase

  •  
  • AMPK

    5′ adenosine monophosphate-activated protein kinase

  •  
  • AST

    aspartate transaminase

  •  
  • CD

    cluster of differentiation

  •  
  • COX-2

    cyclo-oxygenase-2

  •  
  • CYP2E1

    cytochrome P450 2E1

  •  
  • DMEM

    Dulbecco's modified Eagle's medium

  •  
  • H&E

    haematoxylin and eosin stain

  •  
  • ICAM-1

    intercellular adhesion molecule 1

  •  
  • IFN

    interferon

  •  
  • IL

    interleukin

  •  
  • iNOS

    inducible nitric oxide synthase

  •  
  • IκB

    inhibitor of NF-κB

  •  
  • LPS

    lipopolysaccharide

  •  
  • MCP-1

    monocyte chemoattractant protein-1

  •  
  • NF-κB

    nuclear transcription factor κB

  •  
  • PPAR

    peroxisome-proliferator-activated receptor

  •  
  • ROS

    reactive oxygen species

  •  
  • RT

    reverse transcription

  •  
  • TNF

    tumour necrosis factor

  •  
  • WT

    wild-type

AUTHOR CONTRIBUTION

Dong Lee and Dae Kim designed experiments, researched data and wrote the manuscript. Chul Hwang contributed to preparation and interpretation of data. Young Jung designed experiments and researched data. Sukgil Song, Sang Han, Youngsoo Kim, Hwan Yoo, Soo Kim and Do Yoon contributed to the interpretation of data and edited the manuscript before submission. Jin Hong designed experiments, analysed and interpreted data and edited the manuscript before submission. All authors approved the final version of the manuscript.

We thank the Laboratory Animal Research Center in Chungbuk National University for technical support.

FUNDING

This research was supported by the National Research Foundation of Korea [grant number 2012R1A2A2A 02008751].

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Author notes

1

These authors contributed equally to this work.