Interleukin-32: A new proinflammatory cytokine involved in hepatitis C virus-related liver inflammation and fibrosis

Authors

  • Alexander R. Moschen,

    1. Christian Doppler Research Laboratory for Gut Inflammation, Innsbruck Medical University, Innsbruck, Austria
    2. Department of Internal Medicine II (Gastroenterology and Hepatology), Innsbruck Medical University, Innsbruck, Austria
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    • These authors contributed equally to this work.

  • Teresa Fritz,

    1. Christian Doppler Research Laboratory for Gut Inflammation, Innsbruck Medical University, Innsbruck, Austria
    2. Department of Internal Medicine II (Gastroenterology and Hepatology), Innsbruck Medical University, Innsbruck, Austria
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    • These authors contributed equally to this work.

  • Andrew D. Clouston,

    1. School of Medicine, Southern Clinical Division, University of Queensland, Princess Alexandra Hospital, Brisbane, QLD, Australia
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  • Ilka Rebhan,

    1. Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany
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  • Oliver Bauhofer,

    1. Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany
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  • Helen D. Barrie,

    1. School of Medicine, Southern Clinical Division, University of Queensland, Princess Alexandra Hospital, Brisbane, QLD, Australia
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  • Elizabeth E. Powell,

    1. School of Medicine, Southern Clinical Division, University of Queensland, Princess Alexandra Hospital, Brisbane, QLD, Australia
    2. Department of Gastroenterology and Hepatology, Princess Alexandra Hospital, Brisbane, QLD, Australia
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  • Soo-Hyun Kim,

    1. Laboratory of Cytokine Immunology, Institute of Biomedical Science and Technology, College of Medicine, Konkuk University, Seoul, Korea South
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  • Charles A. Dinarello,

    1. Department of Medicine, Division of Infectious Diseases, University of Colorado, Aurora, CO, USA
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  • Ralf Bartenschlager,

    1. Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany
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  • Julie R. Jonsson,

    1. School of Medicine, Southern Clinical Division, University of Queensland, Princess Alexandra Hospital, Brisbane, QLD, Australia
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  • Herbert Tilg

    Corresponding author
    1. Christian Doppler Research Laboratory for Gut Inflammation, Innsbruck Medical University, Innsbruck, Austria
    2. Department of Internal Medicine II (Gastroenterology and Hepatology), Innsbruck Medical University, Innsbruck, Austria
    • Christian Doppler Research Laboratory for Gut Inflammation, Innsbruck Medical University, Anichstraβe 35, 6020 Innsbruck, Austria
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    • fax: +43 512 504 67 23374


  • Potential conflict of interest: Nothing to report.

  • Grant support was provided by the Christian-Doppler Research Society (to H.T.), National Health and Medical Research Council Australia (to E.E.P., A.D.C., J.R.J.), Sasakawa Foundation (Royal Children's Hospital, Brisbane, to J.R.J.), the Sonderforschungsbereich TRR77 (Teilprojekt A1 to R.B.), and by the NIH Grant AI-15614 (to C.A.D.).

Abstract

Interleukin 32 (IL-32) is a recently described proinflammatory cytokine that activates p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB), thereby inducing proinflammatory cytokines such as IL-1β and tumor necrosis factor alpha (TNF-α). We investigated the role of IL-32 in patients with chronic hepatitis C virus (HCV) infection. Steady-state hepatic messenger RNA (mRNA) levels of IL-32 were determined in a cohort of 90 subjects; anti-IL-32 staining was used in a second cohort of 132 consecutive untreated chronic HCV patients. Correlations with histological features of steatosis, inflammation, and fibrosis were made. In vitro, endogenous IL-32 in monocytes and in the human hepatoma cell line Huh-7.5 were examined. The effects of IL-32-overexpression and IL-32-silencing on HCV replication were studied using HCV luciferase reporter viruses. There were highly significant positive associations between hepatic IL-32 mRNA expression and liver steatosis, inflammation, fibrosis, smooth muscle actin (SMA) area, and serum alanine aminotransferase (ALT) levels. IL-32 protein expression was positively associated with portal inflammation, SMA area, and ALT. In vitro, IL-1β and TNF-α significantly induced IL-32 expression in human Huh-7.5 cells. Alone, stimulation with interferon alpha (IFN-α) did not induce IL-32 expression in Huh-7.5. However, IFN-α exerted a significant additive effect on TNF-α-induced but not IL-1β-induced IL-32 expression, particularly in CD14+ monocytes. This effect was dependent both on NF-κB and Jak/STAT signaling. Viral infection of Huh-7.5 cells resulted in a significant (11-fold) induction of IL-32 mRNA expression. However, modulation of IL-32 in Huh-7.5 cells by overexpression or silencing did not influence HCV virus replication as determined by luciferase assays. Conclusion: IL-32 is a novel proinflammatory cytokine involved in HCV-associated liver inflammation/fibrosis. IL-32 is expressed by human hepatocytes and hepatoma cells and its expression is regulated by proinflammatory stimuli. (HEPATOLOGY 2011;)

Hepatitis C virus (HCV) infection is one of the leading causes of chronic liver disease, affecting more than 170 million people worldwide. Chronic HCV infection is a major cause of endstage liver disease resulting in liver cirrhosis and hepatocellular carcinoma. HCV-related liver cirrhosis has become a leading indication for liver transplantation in the Western world.1

As part of the body's antiviral strategy, HCV induces an early innate immune response comprising the induction of antiviral and immunoregulatory cytokines that are vital for the determination of disease outcomes.2 However, most often HCV infection becomes persistent and causes acute and chronic liver disease.3, 4 Whereas acute hepatitis is characterized by activation of cytotoxic CD8+ T cells,5 viral clearance is associated with the entry and accumulation of HCV-specific interferon (IFN)-γ-producing T cells with induction of IFN-γ-induced genes in the liver.6 These comprise proinflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha (TNF-α).7 TNF-α plays a key role in bystander killing of infiltrating cytotoxic T lymphocytes, thereby contributing to the immunopathology associated with HCV.8

In 1992, Dahl et al.9 reported the expression of a novel gene in peripheral cells of patients receiving high doses of IL-2 and cloned the complementary DNA (cDNA) from a human natural killer (NK) cell library; the cDNA was designated NK4. However, for the next 12 years the function of NK4 remained unknown. Kim et al.10 expressed the NK4 cDNA and purified the recombinant protein in 2005. Recombinant NK4 exhibited properties of a proinflammatory cytokine inducing IL-1β and TNF-α in human monocytic cells and they renamed NK4 as IL-32. Subsequently, IL-32 was reported to be involved in several chronic inflammatory diseases including Crohn's disease, ulcerative colitis,11, 12 and rheumatoid arthritis.13 Other studies demonstrated its proinflammatory role in various disease models. IL-32 expression is increased in lung tissue of patients with chronic obstructive pulmonary disease (COPD).14 In that study, IL-32 staining correlated with that of TNF-α and with the degree of airflow obstruction. Two recent studies demonstrated that IL-32 is expressed and functional as a proinflammatory mediator in human vascular endothelial cells.15, 16 IL-32 propagated vascular inflammation, and endothelial expression of IL-32β in transgenic mice promoted inflammation and worsened sepsis.16 Moreover, IL-32 has been implicated in infectious diseases such as mycobacterium tuberculosis, influenza A virus, and human immunodeficiency virus (HIV)-1 infections.17-20 Importantly, IL-32 was reported to suppress HIV-1 replication.19, 20 IL-32 is not only induced during infection with Mycobacterium tuberculosis,17 but as recently demonstrated might also play a role in the host defense against this bacterium.21

Thus, the aim of this study was to evaluate the role of IL-32 in chronic HCV infection. Specifically, we examined IL-32 in patients with untreated chronic HCV infection to assess any association with viral load and liver fibrosis, steatosis, or inflammation. In vitro, we determined the impact of proinflammatory cytokines and type I interferon on endogenous IL-32 expression in human hepatocytes. Moreover, using HCV luciferase reporter viruses we investigated (1) whether HCV infection affects expression of IL-32 in vitro and (2) studied the influence of IL-32 on HCV replication.

Abbreviations

ALT, alanine aminotransferase; BMI, body mass index; COX, cyclooxygenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HCV, hepatitis C virus; HIV, human immunodeficiency virus; IFN, interferon; IL, interleukin; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor kappa B; PBMC, peripheral blood mononuclear cells; SMA, smooth muscle actin; TNF, tumor necrosis factor.

Materials and Methods

Subjects and Clinical Assessment.

In the present study two cohorts of 90 (mRNA study) and 132 (immunohistochemistry study) Caucasian patients with chronic HCV infection were included. Each subject had undergone a percutaneous liver biopsy at the Princess Alexandra Hospital, Brisbane, Australia. Some patients from this cohort have been the subject of earlier reports.22-24 The study was approved by the Princess Alexandra Hospital Research Ethics Committee and the University of Queensland Medical Research Ethics Committee and written, informed consent was obtained from each study patient. Chronic HCV was diagnosed by standard serological assays and abnormal serum aminotransferase levels for at least 6 months. All patients were positive for HCV antibody by the third-generation enzyme-linked immunosorbent assay (ELISA) (Abbott Laboratories, North Chicago, IL) with infection confirmed by detection of circulating HCV RNA by polymerase chain reaction (PCR) using the Amplicor HCV assay (Roche, Branchburg, NJ). Viral genotyping was performed using the Inno-Lipa HCV II assay (Innogenetics, Zwijnaarde, Belgium). Patients with other forms of chronic liver disease or antibodies to HIV were not considered for the analysis. Details about alcohol intake (g/day) during the preceding 12 months and prior to the last 12 months were obtained from all patients at the time of liver biopsy. Serum was collected at the time of liver biopsy following an overnight fast for 8-10 hours. Routine biochemical tests were performed using a Hitachi 747-100 Analyser (Roche, Castle Hill, New South Wales, Australia).

Sample Collection and Histopathological Assessment and Scoring.

After liver biopsy, a 2-3 mm segment of the biopsy was immediately frozen in liquid nitrogen and stored at −80°C until RNA extraction. The remaining biopsy core was fixed in 10% buffered formalin and embedded in paraffin. The sections were analyzed by an experienced hepatopathologist (A.C.) in a blinded fashion. The degree of inflammation was graded according to the method of Ishak et al.,25 and fibrosis was staged according to the method of Scheuer and colleagues.26 Steatosis was graded as follows: 0 (<5% hepatocytes affected); 1, (5%-33% of hepatocytes affected); 2, (34%-66% of hepatocytes affected); or 3, (>66% of hepatocytes affected). Staining and quantification of hepatic smooth muscle actin (SMA), a marker for activated hepatic stellate cells, was as described.27

Immunohistochemistry and Immunofluorescence Double Staining.

Formalin-fixed paraffin-embedded liver biopsy specimens (n = 132) were used for immunohistochemical studies using a polyclonal anti-IL-32 antibody to human IL-32 as described.13 For some experiments liver specimens were obtained from patients undergoing orthotopic liver transplantation for hepatitis B virus, primary sclerosing cholangitis, autoimmune hepatitis, or alcoholic liver disease-related cirrhosis (n = 3 per group). Healthy liver biopsies from two patients with metastatic liver disease undergoing liver resection served as controls. Immunofluorescence double staining was performed on OCT-embedded cryosections. Methodical details are described in the Supporting Experimental Procedures.

Determination of IL-32 mRNA Levels.

IL-32 mRNA levels were assessed by quantitative real-time PCR assays using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18S RNA as the housekeeping genes. Details are given in the Supporting Experimental Procedures.

In Vitro Experiments.

For in vitro experiments the human hepatocellular carcinoma cell line Huh-7.5 was used.28 Hep3B hepatoma cells (HB-8064, American Type Culture Collection) were used for confirmation experiments. Isolation of CD14+ monocytes was performed as described.29 Please see Supporting Experimental Procedures for cell culture details.

Western Blot.

Whole cell lysate was prepared using M-PER mammalian protein extraction reagent according to the manufacturer's instructions (Pierce, Rockford, IL). Please see Supporting Experimental Procedures for details.

IL-32 Variants Overexpression and RNA Interference.

For HCV replication assays, IL-32β (Accession No. NM_001012631) and γ (Accession No. NM_001012635) variants were overexpressed using pTarget mammalian expression vectors (Promega, Madison, WI). Production of the γ-variant was as described.10 Vector efficiency is demonstrated in Fig. 5A. IL-32 was silenced using specific small interfering RNAs (siRNAs). Silencing capacity is demonstrated in Fig. 5B. The HCV-specific siRNA HCV321 (sequence: AGGUCUCGUA GACCGUGCA) was purchased from MWG.30 Please see Supporting Experimental Procedures for details.

Luciferase Assays.

The construction of a bicistronic reporter virus carrying a firefly-luciferase reporter gene (pFK-Luc-Jc1) has been reported.31 Luciferase reporter gene activity was quantified to determine transient HCV RNA replication. Production of cell culture-derived HCV is reported in the Supporting Experimental Procedures.

Statistical Analysis.

Continuous normally distributed variables are represented graphically as mean ± standard error of the mean (SEM). Age, current or past alcohol consumption are summarized by the median followed by range as indicated. To compare the means between groups, analysis of variance (ANOVA) with post-hoc Bonferroni was performed. To determine differences between groups not normally distributed, medians were compared using Kruskal-Wallis analysis of variance (ANOVA) or the Mann-Whitney U test. The degree of association between variables was assessed using Spearman's nonparametric correlation. All statistical analyses were carried out using the PASW Statistics 17.0 software package (SPSS, Chicago, IL) and graphical illustrations were prepared using GraphPad Prism v. 5 (http://www.graphpad.com/).

Results

Patient Characteristics

Cohort 1: Steady-State mRNA Levels.

The demographic, biochemical, metabolic, and histological characteristics of the 90 study patients with chronic HCV infection used for mRNA studies are summarized in Table 1. The body mass index (BMI) ranged from 18.9 to 40.6 kg/m2. In all, 36% of patients had BMI >25 kg/m2 and 16% had BMI >30 kg/m2. Current alcohol use was above recommended guidelines (>20 g/day for females, >30 g/day for males) for 14% of patients, whereas past ethanol use was above recommended guidelines for 53% of patients. Fasting serum insulin was <15 mU/L for 90% of patients and fasting serum glucose was ≤110 mg/dL for 96% of patients. Serum alanine aminotransferase (ALT) was greater than 2× the reference range (19 U/L for females, 30 U/L for males) for 88% of patients.

Table 1. Demographic, Histological, and Biochemical Characteristics of HCV Study Patients
CharacteristicmRNA Expression Study Patients n = 90Immunohistochemistry Study Patients n = 132
  • Data are presented as mean ± SEM, or median (range); ALT, alanine aminotransferase.

  • *

    Interface hepatitis and lobular inflammation was unavailable for five patients in the immunohistochemistry cohort.

Gender (m/f)58/3286/46
Age (range)40.3 (19.1-58.2)40.9 (21.4-58.2)
Current ethanol (g/day)1.0 (0-120)1.4 (0-120)
Past ethanol (g/day)30 (0-500)26 (0-400)
Fibrosis (stage 0,1,2,3,4)10/42/25/5/88/49/39/15/21
Steatosis (grade 0,1,2,3)37/32/17/452/42/24/14
Interface hepatitis (grade 0,1,2,3,4)*15/49/17/9/018/55/41/13/0
Lobular inflammation (grade 0,1,2,3,4)*2/52/29/6/12/61/47/15/2
Portal inflammation (grade 0,1,2,3,4)0/49/37/4/00/60/64/3/0
Total inflammation (grade 1-3, 4-6, 7-9)37/45/843/72/17
ALT (Units/L)124 ± 9.1129 ± 9.7

Cohort 2: Immunohistochemistry.

A second cohort of patients with chronic HCV infection was studied for analysis of IL-32 by specific immunohistochemistry. The demographic, biochemical, metabolic, and histological parameters of the 132 patients are summarized in Table 1. Parameters did not differ significantly from the patients in the gene expression study (Cohort 1). BMI ranged from 16.9 to 42 kg/m2. In all, 39% of patients had BMI >25 kg/m2 and 17% had BMI >30 kg/m2. Current alcohol use was above the recommended guidelines for 11% of patients, whereas past ethanol use was above the guidelines for 41% of patients. Fasting serum insulin was <15 mU/L for 84% of patients and fasting serum glucose was ≤110 mg/dL for 97% of patients. Serum ALT was greater than 2× the reference range (19 U/L for females, 30 U/L for males) for 85% of patients.

Association of Hepatic IL-32 mRNA Expression with Histological, Demographic, and Biochemical Parameters

Steady-state levels of hepatic IL-32 mRNA were readily detected in each patient sample, with a median Ct of 21.1 (range, 18.3-25.1). There were significant correlations between steady-state hepatic IL-32 mRNA levels and total inflammation score (Fig. 1A) and interface hepatitis (Fig. 1B) but not with grade of lobular inflammation (Fig. 1C) or portal inflammation (Fig. 1D). There was also a significant association between IL-32 mRNA expression and the stage of fibrosis (according to Scheuer and colleagues26) (rs = 0.412, P < 0.001) as well as fibrosis rate (Scheuer fibrosis divided by duration of infection) (rs = 0.383, P < 0.001). As shown in Fig. 1E, hepatic IL-32 expression was significantly elevated in patients with severe fibrosis or liver cirrhosis compared with patients without or with mild fibrosis, whereas patients with moderate liver fibrosis showed intermediate IL-32 expression levels. Of note, IL-32 mRNA (rs = 0.272, P < 0.05, Fig. 2B) was significantly correlated with smooth muscle actin as a marker for activated hepatic stellate cells.27 The grade of liver steatosis was significantly positively associated with IL-32 mRNA levels (rs = 0.360, P < 0.01). Patients with steatosis exceeding 30% (grades 2 and 3) showed significantly higher IL-32 expression compared to patients with grade 0 (<5% of hepatocytes) steatosis (Fig. 1F).

Figure 1.

Expression of hepatic IL-32 mRNA and correlation with liver histology. The relationship between steady-state hepatic IL-32 mRNA levels and (A) total inflammation score, (B) interface hepatitis, (C) grade of lobular inflammation, (D) portal inflammation, (E) stage of liver fibrosis, and (F) grade of liver steatosis is demonstrated (Cohort 1; 90 patients). Data are normalized to the first category for each variable and expressed as mean ± SEM. P-values were determined by ANOVA with post-hoc Bonferroni. *P < 0.05 or **P < 0.01 always compared with the first category.

Figure 2.

Expression of hepatic IL-32 mRNA and correlation with various inflammatory and biochemical parameters. Correlation of hepatic IL-32 mRNA levels with (A) hepatic TNF-α expression, (B) hepatic SMA area as a marker for activated hepatic stellate cells, (C) serum ALT levels, and with liver albumin expression (D). Correlation coefficients and level of statistical significance are indicated.

No association was observed between hepatic IL-32 mRNA expression and age, gender, BMI, and current or past alcohol intake. Furthermore, no relationship was found between IL-32 mRNA levels and viral load (available for 38 patients) or viral genotype (data not shown). Hepatic IL-32 mRNA levels were positively correlated with TNF-α mRNA expression (rs = 0.501, P < 0.001; Fig. 2A). Hepatic IL-32 mRNA levels were also significantly associated with serum ALT levels (rs = 0.318, P < 0.01; Fig. 2C, Table 2A). IL-32 mRNA was significantly negatively related with hepatic albumin expression (rs = −0.309, P < 0.05; Fig. 2D, Table 2A).

Table 2A. Relationship of Hepatic IL-32 mRNA with Histological and Clinical Parameters in Hepatitis C Patients
VariableSpearman Correlation Coefficient (rs)P-value
  1. *mRNA expression.

  2. SMA, smooth muscle actin (area); ALT, aspartate aminotransferase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Scheuer fibrosis0.412<0.001
Steatosis0.3600.004
Ishak A0.408<0.001
Ishak A/B/C/D0.367<0.001
SMA (area)0.2720.012
ALT (Units/L)0.3180.002
Albumin/GAPDH*-0.3090.012

Association of Hepatic IL-32 Protein Expression with Histological, Demographic, and Biochemical Parameters

Immunoperoxidase staining revealed expression of IL-32 in nearly all hepatocytes (Fig. 3A-C), although in most patients' samples the intensity of staining was moderate in degree. Variable weaker staining was seen in bile duct epithelium but also in cells of the portal inflammatory infiltrate (Fig. 3C). There was minor staining of lobular inflammatory cells in areas of lobular hepatitis. IL-32 was not observed in Kupffer cells. No association was observed between hepatic intensity of IL-32 staining and age, gender, BMI, and current or past alcohol intake. Notably, a highly significant positive relationship was observed between IL-32 positivity and viral genotype for both hepatocyte (rs = 0.325, P < 0.001) and portal (rs = 0.177, P < 0.05) IL-32 expression. Hepatocyte IL-32 staining was significantly stronger in genotype 3 (n = 40) compared with genotype 1 (n = 86) patients as determined by ANOVA with post-hoc Bonferroni (genotype 1: 2.11 ± 0.05, genotype 3: 2.47 ± 0.08, P < 0.001, data not shown). Moreover, portal but not hepatic IL-32 positivity was significantly associated with serum ALT (rs = 0.250, P < 0.05). Immunohistochemical association studies are summarized in Table 2B.

Figure 3.

Immunohistochemical staining of IL-32 in liver biopsy sections. Immunohistochemical stainings were performed in cohort 2 (132 patients). IL-32 positivity was found in all hepatocytes (A-C). Weaker staining was seen in bile duct epithelium (C, open arrowheads) and in some cells of the portal inflammatory infiltrate (C, black arrows). There is a significant relationship of both portal (D) and hepatocyte (E) IL-32 positivity with hepatic steatosis. Portal IL-32 positivity was associated with the grade of portal inflammation (F). Data are expressed as mean ± SEM. *P < 0.05 or **P < 0.01 always compared with the first category.

Table 2B. Relationship of IL-32 Protein Expression as Characterized by Positivity in Immunohistochemistry with Histological and Clinical Parameters in Hepatitis C Patients
Hepatocyte IL-32 Immunoreactivity withSpearman Correlation Coefficient (rs)P-value
Viral genotype0.325<0.001
Steatosis0.2480.004
Glucose (mg/dl)−0.2090.029
Portal IL-32 Immunoreactivity withSpearman Correlation Coefficient (rs)P-value
Viral genotype0.1770.043
Steatosis0.2530.003
Scheuer fibrosis0.1670.049
Ishak D0.281<0.001
SMA0.2290.048
ALT0.2500.025

Portal staining for IL-32 was weakly but significantly associated with the stage of fibrosis (rs = 0.175, P < 0.05). Again, as seen for mRNA levels, both portal (Fig. 3D) and hepatic (Fig. 3E) IL-32 staining was significantly more intense in samples from patients with steatosis exceeding 30% (grades 2 and 3) compared with patients with grade 0 (<5% of hepatocytes) steatosis. There was also a significant association between portal IL-32 protein expression and grade of portal inflammation (according to Ishak et al.25) (rs = 0.281, P < 0.001). Portal IL-32 staining was significantly greater in patients with grade 3 compared with patients with grade 1 portal inflammation (Fig. 3F). Moreover, portal IL-32 positivity was significantly associated with SMA (rs = 0.229, P < 0.05).

As shown in Supporting Fig. 1, IL-32 positivity was enhanced in various chronic liver diseases such as alcoholic cirrhosis, primary biliary cirrhosis, autoimmune hepatitis, and HBV infection compared with normal liver tissue. Cellular sources of IL-32 were further confirmed by immunofluorescence double labeling. As expected from immunohistochemical studies, IL-32 colocalized with hepatocytes and sinusoidal endothelial cell, but not with Kupffer cells and hepatic stellate cells (Supporting Fig. 2).

IL-32 Is Constitutively Expressed in Huh-7.5 Human Hepatoma Cells and Is Up-regulated by IL-1β and TNF-α

Because IL-32 was readily detected in human hepatocytes by immunohistochemistry, we next examined the regulation of endogenous IL-32 in human Huh-7.5 hepatoma cells. Although steady-state mRNA levels coding for IL-32 were constitutively present in Huh-7.5 cells, there was a significant increase after stimulation with recombinant human IL-1β or TNF-α for 6 hours (Fig. 4A). Combination therapy of IFN-α with ribavirin is a hallmark in the treatment of chronic hepatitis C.32, 33 We therefore determined whether IFN-α affected endogenous IL-32 gene expression. As shown in Fig. 4A, the addition of 1,000 U/mL of IFN-α did not change IL-32 expression. A concentration of 2,500 U/mL of IFN-α also had no effect (data not shown). Whereas IFN-α did not affect IL-1β-induced IL-32 expression (Fig. 4A), the combination of TNF-α and IFN-α resulted in a significant synergistic induction of IL-32 in Huh-7.5 cells. This effect was critically dependent on NF-κB. Inhibition of NF-κB signaling (by BAY 11-7082) but not Jak/STAT signaling (by Jak Inhibitor I) completely abrogated IL-32 induction after stimulation with TNF-α alone or in combination with IFN-α (Fig. 4B). Similar observations were made after 24 hours. We also observed comparable data in Hep3B cells, another human hepatoma cell line (data not shown).

Figure 4.

Regulation of IL-32 gene expression in Huh-7.5 cells and monocytes. IL-32 is produced constitutively by the human hepatoma cell line Huh-7.5. One thousand U/mL of recombinant human IFN-α did not affect IL-32 expression. Stimulation with 1 ng/mL IL-1β or 25 ng/mL TNF-α significantly induced IL-32 mRNA expression after 6 hours. Costimulation of Huh-7.5 cells with TNF-α and IFN-α resulted in significantly stronger IL-32 induction as compared to stimulation with TNF-α alone (A). Blocking NF-κB using the specific pathway inhibitor BAY 11-7082 completely abrogated TNF-α-induced IL-32 induction in Huh-7.5 hepatocytes, whereas Jak Inhibitor I had no effect (B). IFN-α did not induce IL-32 expression in primary CD14+ monocytes. There was again a synergistic effect of IFN-α on TNF-induced IL-32 expression in human monocytes (C). In contrast to hepatocytes IL-32 induction in CD14+ monocytes was dependent on both NF-κB and Jak/STAT signaling (D). mRNA results were confirmed by western blot analysis (shown for monocytes, E).

CD14+ monocytes were also stimulated with TNF-α alone or in combination with IFN-α. Again, IFN-α did not affect IL-32 expression in CD14+ monocytes. However, as shown in Fig. 4C, the combination of TNF-α with IFN-α resulted in a highly synergistic induction of IL-32. For example, after 12 hours there was a 38-fold increase (TNF-α plus IFN-α) compared with a 5.7-fold increase with TNF-α alone. In contrast to hepatocytes, in CD14+ monocytes IL-32 induction was dependent on both NF-κB and Jak/STAT signaling, as demonstrated by inhibitor experiments (Fig. 4D). IL-32 protein levels were similarly elevated in immunoblot analysis (Fig. 4E).

Hepatitis C Replication Is Not Modulated by IL-32, but Viral Infection Stimulates IL-32 Expression

Overexpression of IL-32.

We next examined a potential antiviral effect of IL-32 on HCV replication. Two different experiments were performed. First, we studied the role of endogenous IL-32 in HCV replication by transfecting Huh-7.5 cells with a control plasmid or plasmids overexpressing either IL-32β or IL-32γ under the control of a cytomegalovirus (CMV) promotor (Fig. 5A). Neither overexpression of IL-32β nor of IL-32γ (Fig. 5C) affected HCV replication as measured by luciferase activity compared with the negative control-transfected cells.

Figure 5.

The impact of hepatocyte IL-32 expression on HCV replication. IL-32β and IL-32γ were transiently overexpressed in Huh-7.5 cells as described in Materials and Methods (A). Replication of Jc1-FLuc was quantified in IL-32 transfected and control transfected cells using a luciferase assay (C). IL-32 was efficiently silenced in Huh-7.5 cells using specific siRNAs (B). Technical aspects can be found in Materials and Methods. Again, HCV replication was determined in silenced and control silenced Huh-7.5 hepatoma cells by luciferase assay (D). (B,C) The mean of three independent experiments each performed in duplicate. Values are normalized to the negative control-transfected cells. IL-32β and IL-32γ were transiently overexpressed in Huh-7.5 cells as described in Materials and Methods. Overexpression (A) and silencing (B) of IL-32 in Huh-7.5 hepatocytes were verified by western blot. Replication of Jc1-FLuc was quantified in IL-32β or IL-32γ-transfected and control-transfected cells using a luciferase assay (C). HCV replication was then determined in IL-32-silenced and control-silenced Huh-7.5 hepatoma cells by luciferase assay (D). (C,D) The mean of three independent experiments, each performed in duplicate. Data are normalized to control-transfected cells. Huh-7.5 cells were inoculated with Jc1 at an MOI of ≈100 TCID50 per cell or mock-treated and efficiency of infection was verified by NS5A staining; nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI); merge means the overlay of nuclei (blue) and NS5A (red) (E). HCV infection induced IL-32 mRNA transcription after 48 hours (F). Data represent mean of two independent experiments each performed in duplicate. Values were normalized to GAPDH mRNA levels and compared with mock-treated cells.

siRNA Studies.

Endogenous IL-32 was silenced using IL-32 siRNAs specific for all IL-32 isoforms. The efficiency of IL-32 siRNA silencing was confirmed by a reduction in the protein levels by immunoblot analysis (Fig. 5B). A scrambled siRNA served as a negative control and siRNA targeting of the viral genome (siHCV321) served as a positive control.30 Silencing of endogenous IL-32 (Fig. 5D) did not affect HCV replication as measured by luciferase activity.

HCV Infection Induces IL-32 Transcription In Vitro.

Although IL-32 did not affect HCV replication, we determined whether viral infection stimulates expression of this cytokine. Huh-7.5 cells were inoculated with Jc1 at a multiplicity of infectivity (MOI) of ≈100 TCID50 (50% tissue culture infective dose) per cell to ensure synchronous infection of all cells in the culture dish. Infection of Huh-7.5 hepatocytes was verified by immunofluorometrical detection of NS5A (Fig. 5E). IL-32 mRNA levels were quantified after 24 and 48 hours (Fig. 5F). After normalization to GAPDH mRNA levels in each sample, IL-32 mRNA levels of HCV-infected cells were compared with mock-treated cells. No significant induction of IL-32 mRNA could be detected at the early timepoint, whereas 48 hours postinfection IL-32 mRNA levels were increased 11.3-fold.

Discussion

In the present study we describe a novel role for IL-32 in patients with chronic HCV infection. The levels of IL-32 mRNA were significantly correlated with hepatic inflammation, liver fibrosis, and steatosis. In addition, we demonstrate that IL-32 is endogenously produced by human hepatocyte cell lines and in primary human blood monocytes and is increased upon stimulation with IL-1β and TNF-α. Furthermore, we show that HCV infection of Huh-7.5 cells significantly increases IL-32 expression. Thus, these observations support a potential role for IL-32 in promoting hepatic inflammation and fibrogenesis in chronic HCV infection.

IL-32 exerts proinflammatory effects in various cell types including epithelial and endothelial cells as well as mononuclear cells.10, 15 Consistent with these reports, we observed a highly significant association between IL-32 expression and hepatic inflammation. IL-32, a major monocyte/macrophage product, stimulates monocytes and macrophages to induce important proinflammatory cytokines (IL-1β, IL-6, and TNFα) and chemokines (IL-8 and MIP-2) by activating the NF-κB and p38 mitogen-activated protein (MAP) kinase pathways. IL-32 is not only involved in host defense against pathogens, but might play a role in various chronic inflammatory diseases as suppression of endogenously IL-32 impairs production of the proinflammatory cytokines TNF-α and IL-1β.34 This cytokine, namely IL-32, contributes to host responses through the induction of other proinflammatory cytokines but also directly affects specific immunity by differentiating monocytes into macrophage-like cells.35 Importantly, IL-32 even reversed granulocyte-macrophage colony-stimulating factor (GM-CSF)/IL-4-induced dendritic cell differentiation to macrophage-like cells, suggesting that it might indeed reflect a key cytokine for macrophage development.35 Apoptotic cell death is a critical mechanism responsible for liver injury in chronic HCV and additionally contributes to hepatic fibrogenesis. IL-32, which is expressed by primary human keratinocytes, is able to modulate keratinocyte apoptosis, as transfection of primary keratinocytes with siRNA to IL-32 significantly reduced keratinocyte apoptosis.36 A proapoptotic effect for IL-32 was also demonstrated in activated T cells and NK cells. IL-32 was highly expressed in T cells undergoing apoptosis, whereas down-regulation of IL-32 prevented apoptosis.37 These data together suggest that IL-32 could contribute by way of various mechanisms to development of inflammation and fibrosis in chronic HCV: (1) by affecting monocyte differentiation and induction of other proinflammatory and profibrogenic cytokines, and (2) by induction of apoptosis, which also might stimulate fibrogenesis. Our observations for a major proinflammatory function of IL-32 in chronic HCV are in accordance with those presented by Joosten et al.13 demonstrating that IL-32 was specifically up-regulated in synovial tissue of patients with rheumatoid arthritis (but not in patients with osteoarthritis) and correlated with markers of systemic and synovial inflammation. In patients with COPD, IL-32 staining of fixed lung tissues correlated with disease severity and the level of TNF-α and MAPK p38 expression, again strongly highlighting the association of IL-32 with inflammation and the expression of other proinflammatory cytokines.14

In primary cultured human endothelial cells from the umbilical veins, IL-32 is constitutively expressed and increases upon stimulation with IL-1β.15 In our study, we observed that IL-32 is also constitutively expressed in hepatoma cell lines and increases upon exposure to IL-1β or TNF-α. Moreover, there is a marked synergistic effect of TNF-α plus IFN-α in increasing IL-32 in these cells which can be efficiently blocked by NF-κB and/or Jak/STAT inhibition. IFN-α is a pleiotropic cytokine that exerts numerous antiviral, antiproliferative, and antiinflammatory functions.38 IFN-α alone did not affect IL-32 expression, even at rather high nonphysiologic concentrations such as 1,000 or 2,500 U/mL, suggesting that such an effect might not be functional in vivo. In contrast, the synergistic effect on TNF-induced IL-32 induction in both hepatocytes and especially in CD14+ monocytes may be clinically relevant because this would result in augmented inflammation in the infected liver of these patients. In mice expressing human IL-32β as a transgene, there is greater inflammation with a second stimulus. In fact, it appears that IL-32β expression in transgenic mice increases lipopolysaccharide (LPS) lethality.16

Very recently, Nold et al.20 reported that recombinant IL-32 controls HIV-1 replication in human peripheral blood mononuclear cells (PBMCs). Mechanistically, the authors demonstrated that the antiviral effect was due to IFN-α because antibody to the type I interferon receptor or a neutralizing soluble type I interferon receptor abrogated IL-32′s antiviral capacity.20 We found that type I interferon modulates TNF-induced IL-32 expression. Therefore, we asked whether IL-32 might affect HCV infection. Of note, IL-32 immunoreactivity was significantly higher in patients infected with HCV genotype 3 compared with patients with HCV genotype 1. Antiviral activity has been reported for several proinflammatory cytokines such as IL-1β, IL-12, and TNF-α, and suppression of these cytokines is a well-known mechanism of HCV immune escape.39-41 Using HCV luciferase reporter viruses, we did not observe any antiviral capacity for IL-32 employing two different experimental models. Importantly, however, we demonstrated that HCV infection of Huh-7.5 cells significantly increased IL-32 expression, suggesting that, indeed, viral infection might be a major trigger for the observed hepatic IL-32 expression in chronic HCV infection.

These data indicate that IL-32 affects several parameters of HCV pathology but itself might not have antiviral properties. Other viral infections such as influenza A virus infection also induce IL-32 expression.18, 42 Influenza virus induced cyclooxygenase (COX)-2-mediated prostaglandin E2 production was suppressed by overexpression of IL-32 but decreased by IL-32-specific siRNA, suggesting a feedback mechanism between IL-32 and COX-2.18 A clear antiviral effect against influenza A virus for IL-32 has not been demonstrated in these studies.

In conclusion, in patients with chronic HCV the presence of IL-32 is associated with severity of steatosis, hepatic inflammation, and liver fibrosis. IL-32 is expressed by hepatocytes and up-regulated upon stimulation with IL-1β or TNF-α as well as HCV infection. Although IL-32 lacks anti-HCV activity at least in a cell culture system, our data suggest that viral infection stimulates expression of this cytokine, thus supporting a role for IL-32 in chronic HCV infection and related pathologies.

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