The signals driving T cell activation in T cell-mediated fulminant hepatitis are not fully understood. In this study, we identify the cytokine IL-27p28/EBI3 as a major pathogenic factor in the ConA model of T cell-mediated hepatitis. We found an up-regulation of hepatic EBI3 and p28 expression and augmented levels of IL-27 in wild-type mice after ConA administration, suggesting a potential pathogenic role of this cytokine in ConA hepatitis. Consistently, IL-27 EBI3-deficient mice were almost completely protected from ConA-induced liver damage. Such protection was associated with reduced levels of IFN-γ and its signaling proteins pSTAT-1 and T-bet. Finally, in vivo blockade of IL-27 function using a soluble IL-27 receptor fusion protein led to reduced pSTAT1 levels and suppression of liver injury. Taken together, these data demonstrate a key pathogenic role of IL-27 in T cell-mediated liver injury. Furthermore, in vivo blockade of IL-27 emerges as a novel potential therapy for T cell-mediated hepatitis.

Interleukin-27 is a member of the IL-6/IL-12-family that consists of EBV-induced gene 3 (EBI3)2 and p28 (1, 2, 3). It is the only known ligand for the gp130/WSX-1 receptor (1). Macrophages and dendritic cells are the main producers of IL-27, and the IL-27 signaling cascade includes activation of Jak1, STAT-1, STAT-3, STAT-4, and STAT-5 in T cells, STAT-1 and STAT-3 in monocytes, and STAT-3 in mast cells (2, 3, 4).

IL-27 may exert both pro- and anti-inflammatory functions (5, 6). It can augment T cell proliferation and enhance synthesis of IFN-γ through the induction of T-bet and the activation of STAT-4 (1). IL-27 can activate the transcription of T-bet independently from STAT-1 (4), and such induction of T-bet appears to be mainly responsible for IL-27-mediated IFN-γ production (5, 7).

In addition to its proinflammatory effects, there is increasing evidence that IL-27 may suppress immune responses (8, 9). In fact, infection of WSX-1-deficient mice with Trypanosoma cruzii resulted in an immune-mediated liver injury most likely mediated by augmented production of IFN-γ and TNF-α by hepatic T cells and NKT cells (10).

ConA was purchased from Sigma-Aldrich. Anti-STAT1, anti-phospho-STAT-1, anti-STAT3, anti-phospho-STAT-3, and anti-phospho-STAT-4 Abs were obtained from Cell Signaling.

EBI3-knockout mice (9) and corresponding wild-type animals on a 129/C57BL/6 background were bred in the Animal Care Facility in Mainz, Germany. The strain has been formally denoted B6;129X1-EBI3tm Birk.

Hepatocytes from EBI3-deficient and control mice were isolated from mouse livers after perfusion with 0.05% collagenase (Roche) as described previously (11). After 1 day of culture, total RNA was isolated as described below.

Livers were perfused through the portal vein with 0.05% collagenase A (Roche), mechanically disrupted, and incubated with 0.05% collagenase IV. The fraction of nonparenchymal cells was recovered by centrifugation steps (2 × 400 rpm and 1 × 1500 rpm). Isolation of CD11c+ cells, CD11b+CD11c cells, CD90+ cells, and CD45 (liver SECs) was done by magnetic cell separation (Miltenyi Biotech) according to the manufacturer’s instructions. To select liver SECs, negatively selected CD11bCD11c cells were incubated with CD45 Microbeads and CD45 cells were seeded in collagen-coated wells.

Total cellular RNA was isolated from cells and organs with RNeasy columns (Qiagen), including DNase I digestion. Quantitative real time PCR analysis for EBI3, p28, WSX-1, gp130, and hypoxanthine phosphoribosyltransferase was performed by using specific Quantitec primer/probe assays (Qiagen).

C57BL/6 mice were killed 4 h after i.v. injection of ConA and total RNA from the liver was isolated by using QIAamp tissue kit (Qiagen). Specific primers were as follows: EBI3, 5′-GAATCATCAAGCCGGACCCT–3′ and 5′-GAATCATCAAGCCGGACCCT–3′; IFN-γ, 5′-ACACTGCATCTTGGCTTTGC-3′ and 5′-CGGATGAGCTCATTGAATGCT-3′.

For detection of liver sWSX mRNA, wild-type mice were hydrodynamically injected with pcDNA-WSX and control pcDNA vectors as described below. After 4 h, total liver RNA was extracted and RT-PCR was performed using following primers: WSX, 5′-CGTGAGCAGTCAAACCCAGA-3′ and 5′-GGGAAGAGGAAGACTGAAGG-3′.

ConA-induced hepatitis was performed as previously described (12, 13, 14). Transaminase levels were measured in the clinical chemistry laboratory at the University of Mainz. Liver specimens of the large anterior lobe were cut to 5-μm-thin slices and then stained with H&E.

Western blotting was performed using anti-STAT-1 Ab, anti-phospho-STAT-1, anti-STAT-3, anti-phospho-STAT-3, or anti-T-bet Ab at 1/1000 dilution at room temperature. HRP-labeled anti-rabbit or anti-goat IgGs (DakoCytomation) at 1/1000 dilution was used as a secondary Ab. An enhanced luminescence kit (Amersham Biotechnology) was used.

Ten micrograms of pcDNA 3.1-sWSX and 10 mg of pcDNA3.1 were diluted in 1.6 ml of PBS and injected into the tails of wild-type mice. The injection took no longer than 10 s, and the apnea period in mice ranged from 4 to 6 s.

Data were compared by Student’s t test for independent events. P < 0.05 was considered statistically significant.

To determine the role of IL-27 in T cell-mediated hepatitis, we analyzed the hepatic expression of the EBI3 subunit of IL-27 upon ConA administration in wild-type mice. Expression levels of EBI3 and p28 were increased in APCs such as CD11b+CD11c cells and CD11c+ cells and to a much lesser extent in CD90+ T cells from the liver 6 h after injection of ConA, whereas hepatocytes and SECs did not reveal an augmented p28/EBI3 expression (Fig. 1,A). Moreover, we found a significant increase of serum IL-27 levels 4 and 8 h after the administration of ConA as compared with control mice (Fig. 1 B), consistent with a potential regulatory role of this cytokine in T cell-mediated liver injury.

FIGURE 1.

Expression of EBI3/p28 and serum levels of IL-27 after injection of ConA in wild-type mice. A, ConA (25 mg/kg) was injected i.v. into C57BL/6 mice. After 6 h liver cells were isolated and subjected to quantitative real-time PCR using EBI3- and p28-specific primers. A strong induction of p28 and EBI3 expression was detected in CD11b+CD11c cells and CD11c+ cells (∗, p < 0.05) Expression levels of p28 and EBI mRNA are shown as x-fold induction compared with cells derived from PBS-treated mice. B, The serum concentration of IL-27 was determined 1, 4, and 8 h upon injection of ConA or PBS, respectively, by a specific IL-27 ELISA. Mean values ± SD from three independent experiments are shown.

FIGURE 1.

Expression of EBI3/p28 and serum levels of IL-27 after injection of ConA in wild-type mice. A, ConA (25 mg/kg) was injected i.v. into C57BL/6 mice. After 6 h liver cells were isolated and subjected to quantitative real-time PCR using EBI3- and p28-specific primers. A strong induction of p28 and EBI3 expression was detected in CD11b+CD11c cells and CD11c+ cells (∗, p < 0.05) Expression levels of p28 and EBI mRNA are shown as x-fold induction compared with cells derived from PBS-treated mice. B, The serum concentration of IL-27 was determined 1, 4, and 8 h upon injection of ConA or PBS, respectively, by a specific IL-27 ELISA. Mean values ± SD from three independent experiments are shown.

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To address the functional role of IL-27 in the development of ConA-induced liver injury, we took advantage of IL-27/EBI3-deficient mice (9). Injection of 25 mg/kg ConA in IL-27/EBI3-deficient mice resulted in significantly lower serum liver values (aspartate aminotransferase (AST) and alanine aminotransferase (ALT)) as compared with those in wild-type mice (p < 0.001) (Fig. 2,A). Levels of AST and ALT in ConA-treated IL-27/EBI3 knockout mice were not increased, whereas wild-type mice revealed significantly increased liver enzymes (Fig. 2,A). Consistently, the histology of liver sections from IL-27/EBI3-deficient mice showed only mild degenerative changes, whereas marked necrosis was observed in ConA-treated wild-type mice upon injection of 25 mg/kg ConA. In contrast to wild-type mice, liver sections of IL-27/EBI3-deficient mice given 10 mg/kg ConA revealed no signs of inflammation or necrosis (Fig. 2 B).

FIGURE 2.

EBI3-deficient mice are protected from severe liver injury upon challenge with ConA. A, Eight hours after injection of 25 mg/kg (n = 16) or 10 mg/kg (n = 12) ConA, blood was taken from EBI3-deficient mice and wild-type mice. Levels of liver enzymes (AST and ALT) from the indicated groups of mice are shown as mean values ± SD from three independent experiments (∗, p < 0.001). B, H&E-stained liver sections of ConA-treated EBI3-deficient mice and wild-type (wt) mice. Representative sections from each group are shown. Administration of PBS in mice served as control. Injection of 10 mg/kg ConA in EBI3-deficient mice did not result in any histopathological changes.

FIGURE 2.

EBI3-deficient mice are protected from severe liver injury upon challenge with ConA. A, Eight hours after injection of 25 mg/kg (n = 16) or 10 mg/kg (n = 12) ConA, blood was taken from EBI3-deficient mice and wild-type mice. Levels of liver enzymes (AST and ALT) from the indicated groups of mice are shown as mean values ± SD from three independent experiments (∗, p < 0.001). B, H&E-stained liver sections of ConA-treated EBI3-deficient mice and wild-type (wt) mice. Representative sections from each group are shown. Administration of PBS in mice served as control. Injection of 10 mg/kg ConA in EBI3-deficient mice did not result in any histopathological changes.

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The IFN-γ signaling pathway plays a key pathogenic role in ConA-induced liver injury because IFN-γ-, STAT-1-, and T-bet-knockout mice are protected from ConA-mediated hepatitis (12, 13). In contrast, activation of the transcription factor STAT-3 by IL-6 results in protection against ConA-mediated hepatitis (14). Interestingly, IL-27 can activate both STAT-1 and STAT-3 in naive CD4 T cells and NK cells (15, 16). Accordingly, we determined the expression of IFN-γ and STAT signaling proteins upon ConA administration in wild-type and IL-27/EBI3 deficient mice. We found a decreased hepatic IFN-γ expression (Fig. 3,A) and reduced STAT-1 activation (Figs. 3, B and C) in ConA-treated IL-27/EBI3 deficient mice as compared with wild-type mice. Furthermore, the STAT-1-dependent T-box transcription factor T-bet was markedly suppressed in the former mice as compared with the latter mice, suggesting that IL-27 induces STAT-1 and T-bet activation in T cell-mediated hepatitis. Finally, we determined the activation levels of STAT-3 and STAT-4. However, analysis of liver lysates from EBI3-deficient mice revealed no differences in STAT-3 expression and phospho-STAT-3/phospho-STAT-4 levels between wild-type and knockout mice (Fig. 3,B). To address the question of in which cell type the changes of IFN-γ-related proteins occur, we isolated CD11b+CD11c cells, CD11c+ cells, CD90+ lymphocytes, and primary hepatocytes from the livers of ConA-treated EBI3-deficient mice and control mice. Decreased phospho-STAT-1 expression was observed in CD90+ cells, CD11b+CD11c cells, CD11c+ cells, and hepatocytes, whereas no reduction in phospho-STAT-1 expression was detected in SECs (Fig. 3 C). Taken together, these data suggest a critical role of IL-27 EBI3 in controlling IFN-γ production and signaling in T cell-mediated hepatitis via STAT-1 and T-bet.

FIGURE 3.

The IFN-γ signaling pathway is down-regulated in ConA-treated EBI3-deficient mice. A, Expression of hepatic IFN-γ-mRNA was analyzed by RT-PCR 4 h after injection of ConA (25 mg/kg ConA). β-Actin expression served as control. A lower expression of IFN-γ-mRNA was detectable in ConA-treated knockout mice as compared with wild-type (wt) mice. B, Levels of STAT-1, T-bet, and STAT-3 proteins and phospho-STAT proteins in liver lysates from EBI3-deficient and wild-type mice after injection of ConA were analyzed by Western blotting. Levels of phospho-STAT-1 and T-bet were lower in EBI3-deficient mice compared with wild-type controls, while levels of STAT-3, pSTAT-3 and pSTAT-4 were virtually unchanged. C, Analysis of phospho-STAT-1 expression was performed in different liver cell types 6 h after administration of ConA (25 mg/kg) using immunoprecipitation. Expression levels were lower in CD11c+ cells, CD11b+ cells, hepatocytes, and CD90+ cells isolated from EBI3-deficient mice when compared with wild-type controls.

FIGURE 3.

The IFN-γ signaling pathway is down-regulated in ConA-treated EBI3-deficient mice. A, Expression of hepatic IFN-γ-mRNA was analyzed by RT-PCR 4 h after injection of ConA (25 mg/kg ConA). β-Actin expression served as control. A lower expression of IFN-γ-mRNA was detectable in ConA-treated knockout mice as compared with wild-type (wt) mice. B, Levels of STAT-1, T-bet, and STAT-3 proteins and phospho-STAT proteins in liver lysates from EBI3-deficient and wild-type mice after injection of ConA were analyzed by Western blotting. Levels of phospho-STAT-1 and T-bet were lower in EBI3-deficient mice compared with wild-type controls, while levels of STAT-3, pSTAT-3 and pSTAT-4 were virtually unchanged. C, Analysis of phospho-STAT-1 expression was performed in different liver cell types 6 h after administration of ConA (25 mg/kg) using immunoprecipitation. Expression levels were lower in CD11c+ cells, CD11b+ cells, hepatocytes, and CD90+ cells isolated from EBI3-deficient mice when compared with wild-type controls.

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To further analyze the functional role of IL-27/EBI3 in the pathogenesis of T cell-mediated liver pathology, we aimed at blocking IL-27 function in vivo. Accordingly, we performed i.v. hydrodynamic injection of a cDNA encoding for a blocking, soluble IL-27 receptor fusion that has been previously shown to block IL-27 function in vivo and in vitro (17). Upon the administration of ConA, the levels of AST and ALT were significantly lower in mice given the plasmid expressing the soluble receptor fusion protein as compared with mice treated with the control vector (Fig. 4,A), indicating that a blockade of IL-27 signal transduction suppresses T cell-mediated hepatitis in vivo. Histological analysis of liver sections revealed fewer pathological changes in sWSX expression vector-treated mice compared with control vector-treated mice (Fig. 4,B). Because the i.v. injection of cDNA for the soluble IL-27 receptor fusion protein resulted in the intrahepatic expression of sWSX mRNA (Fig. 4,C), our data are consistent with a model in which sWSX is expressed upon hydrodynamic injection followed by the suppression of ConA-mediated hepatitis in vivo. Consistently, Western blot analysis revealed lower levels of phospho-STAT-1 and T-bet expression. In contrast, no changes in phospho-STAT-4 levels were noted, suggesting that STAT-4 can be activated by other cytokines in the absence of IL-27 (Fig. 4 D).

FIGURE 4.

Blockade of IL-27 attenuates ConA-induced liver pathology. A, pcDNA-sWSX or an empty control vector wsd injected in wild-type mice (5–10 mice per group). Twelve hours later ConA (25 mg/kg) was injected. After an additional 8 h, the levels of AST and ALT were analyzed. A significant protection against ConA-mediated hepatitis was found in mice treated with vector DNA encoding the soluble IL-27 receptor fusion protein as compared with the control groups (p < 0.01). B, Histopathology of the livers from wild-type mice treated with sWSX vector DNA plus ConA and control vector DNA plus ConA, respectively, after staining with H&E. Representative sections are shown. C, Expression of intrahepatic sWSX cDNA after hydrodynamic injection. Four hours after hydrodynamic injection of pcDNA-WSX or pcDNA control vector in wild-type mice, livers were removed, mRNA was isolated, and sWSX-specific RT-PCR was performed. Levels of actin expression served as controls. D, Levels of hepatic pSTAT-1, T-bet, and pSTAT-4 expression in wild-type mice injected with sWSX cDNA plus ConA and control cDNA plus ConA were determined by Western blot analysis. E, Expression of gp130 mRNA and WSX mRNA in different liver cell types derived from EBI3-deficient mice and control mice was quantified by real-time PCR in the indicated hepatic cell lines. Expression levels in EBI3-deficient mice are shown as x-fold induction compared with control wild-type mice.

FIGURE 4.

Blockade of IL-27 attenuates ConA-induced liver pathology. A, pcDNA-sWSX or an empty control vector wsd injected in wild-type mice (5–10 mice per group). Twelve hours later ConA (25 mg/kg) was injected. After an additional 8 h, the levels of AST and ALT were analyzed. A significant protection against ConA-mediated hepatitis was found in mice treated with vector DNA encoding the soluble IL-27 receptor fusion protein as compared with the control groups (p < 0.01). B, Histopathology of the livers from wild-type mice treated with sWSX vector DNA plus ConA and control vector DNA plus ConA, respectively, after staining with H&E. Representative sections are shown. C, Expression of intrahepatic sWSX cDNA after hydrodynamic injection. Four hours after hydrodynamic injection of pcDNA-WSX or pcDNA control vector in wild-type mice, livers were removed, mRNA was isolated, and sWSX-specific RT-PCR was performed. Levels of actin expression served as controls. D, Levels of hepatic pSTAT-1, T-bet, and pSTAT-4 expression in wild-type mice injected with sWSX cDNA plus ConA and control cDNA plus ConA were determined by Western blot analysis. E, Expression of gp130 mRNA and WSX mRNA in different liver cell types derived from EBI3-deficient mice and control mice was quantified by real-time PCR in the indicated hepatic cell lines. Expression levels in EBI3-deficient mice are shown as x-fold induction compared with control wild-type mice.

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Finally, we analyzed the expression of WSX and gp130 on different liver cell types using real-time PCR. We detected no significant differences in cellular WSX and gp130 expression between EBI3-deficient and wild-type mice (Fig. 4 E), suggesting that both EBI3-deficient and wild-type cells in the liver are susceptible to IL-27-dependent activation.

The signals driving T cell activation in the ConA-mediated model of hepatitis are not fully understood. In this study, we have shown a key role of IL-27 in controlling T cell-mediated liver injury in this model. IL-27 is rapidly produced by dendritic cells and macrophages in vivo (1), and its serum levels were significantly induced within 4 h after ConA administration. Furthermore, IL-27/EBI3-deficient mice showed reduced IFN-γ production and STAT1/T-bet activation in the ConA model, suggesting that IL-27 may act very early in Th1-mediated immunity in T cell-mediated hepatitis.

Mice deficient in IL-27/EBI3 were markedly protected against ConA-induced liver injury, whereas WSX-1-knockout mice showed augmented ConA hepatitis (18), suggesting marked differences in the phenotype between these mice. Indeed, previous studies have identified differences between IL-27/EBI3- and WSX-1-deficient mice. For instance, Yoshida and colleagues demonstrated by using WSX-1-deficient mice that WSX-1 is required for the normal production of IFN-γ by primary naive CD4+ T cells (5). In contrast, EBI3-deficient T cells showed diminished IL-4 and augmented IFN-γ synthesis (9). Although the reason for these differences is not entirely clear, our data unequivocally identify IL-27 as a central pathogenic factor in the ConA model of T cell-mediated liver injury. Furthermore, because the blockade of IL-27 using an IL-27 receptor fusion protein suppressed ConA-mediated hepatitis in vivo, the targeting of IL-27 emerges as a potential novel approach for therapy of T cell-mediated hepatitis.

We thank Sonja Klein for excellent technical assistance.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

2

Abbreviations used in this paper: EBI3, EBV-induced gene 3; ALT, alanine aminotransferase; AST, aspartate aminotransferase; SEC, sinusoidal endothelial cell; sWSX, soluble WSX.

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