Elsevier

Clinical Immunology

Volume 146, Issue 1, January 2013, Pages 15-25
Clinical Immunology

TNF-α is essential in the induction of fatal autoimmune hepatitis in mice through upregulation of hepatic CCL20 expression

https://doi.org/10.1016/j.clim.2012.10.008Get rights and content

Abstract

It is unclear what roles TNF-α has in the development of autoimmune hepatitis (AIH) and whether AIH is responsive to anti-TNF-α. We recently developed a mouse model of fatal AIH that develops in PD-1-deficient mice thymectomized three days after birth, finding that CCR6-CCL20 axis-dependent migration of dysregulated splenic T cells is crucial to induce AIH. In this study, we show the indispensable role of TNF-α in the development of AIH. Administering anti-TNF-α prevented the induction, but treatment by anti-TNF-α after the induction did not suppress progression. Administering anti-TNF-α did not prevent splenic T-cell activation, but did suppress hepatic CCL20 expression. In contrast, administering anti-CCL20 suppressed AIH but not elevated serum TNF-α levels. TNF-α stimulation enhanced CCL20 expression in hepatocytes. These findings suggest that TNF-α is essential in the induction of AIH through upregulation of hepatic CCL20 expression, which allows migration of dysregulated splenic T cells.

Highlights

► It is unclear what roles proinflammatory TNF-α has in the development of AIH. ► It is unknown whether AIH is responsive to TNF-α antagonists. ► Anti–TNF-α suppressed hepatic CCL20 expression and prevented induction of AIH. ► Anti–TNF-α treatment did not significantly suppress progression of fatal AIH. ► TNF-α is essential in the induction of AIH thorough upregulation of hepatic CCL20 expression.

Introduction

Dysregulated production of tumor necrosis factor (TNF)-α is involved in the pathology of various immune-mediated inflammatory diseases, including inflammatory bowel diseases and rheumatoid arthritis in humans [1], [2], [3], [4]. In the liver, TNF-α directly and indirectly induces cell death of hepatocytes, whereas it can mediate production of inflammatory mediators, hepatocyte proliferation, and liver regeneration [5]. In human diseases, TNF-α is involved in the pathophysiology of viral hepatitis, alcoholic liver disease, nonalcoholic fatty liver disease, and ischemia-reperfusion injury [5]. In addition, serum TNF-α levels were elevated in untreated children with type 1 autoimmune hepatitis (AIH) in comparison with those in healthy controls [6]. A polymorphism at position 308 in a promoter region of TNF-α has been reported to be associated with severity of AIH type 1 in Europe and North America [7], [8]. However, it is unclear whether TNF-α is critical in the development of AIH or whether TNF antagonists have therapeutic efficacies for AIH in humans.

Several mouse models of AIH have demonstrated the roles of cytokines, costimulatory molecules, T-cell subsets, and autoantigens in the development of AIH. Gorham et al. reported that BALB/c background TGF-β1 deficient mice develop fatal hepatitis characterized by massive CD4+ T cell infiltration [9]. In this model, interferon (IFN)-γ produced by CD4+ T cells drove lethal hepatic damage [10]. Mice deficient in B and T lymphocyte attenuator (BTLA), negative costimulatory molecules expressed on lymphocytes, developed AIH-like disease with hypergammaglobulinemia and production of anti-nuclear antibodies [11]. In addition, mice immunizing human liver autoantigens and mice infected with adenovirus Ad5 expressing human cytochrome P450 2D6 developed persistent immune-mediated hepatitis [12]. Mice vaccinated with dendritic cells loaded with liver antigens and subsequently administered IL-12 also developed persistent immune mediated hepatitis [13]. Furthermore, influenza virus hemagglutinin-specific T-cell receptor transgenic mice further expressing the hemagglutinin specifically in the liver spontaneously developed chronic autoimmune-mediated hepatitis [14]. However, in these models, the roles of TNF-α in the development of hepatitis are not clear.

Concanavalin A (Con A)-induced acute hepatic injury, associated with activation of NKT cells and T cells, is considered to be an experimental model of human AIH [15]. A single intravenous injection of Con A into mice rapidly induces injury of hepatocytes together with increased serum levels of TNF-α [16]. Although one report demonstrated that neutralization of TNF-α by anti-TNF-α significantly suppressed Con A-induced hepatic injury, Tagawa et al. showed that hepatocytes in TNF-α-deficient mice were severely injured by Con A to levels similar to wild-type mice [17]. Thus, an essential role of TNF-α in inducing Con A-induced acute hepatic injury is still controversial.

To clarify mechanisms involved in the development of AIH, we recently developed a new mouse model of fatal AIH [18], [19]. Neither programmed cell death 1-deficient mice (PD-1−/− mice) nor BALB/c mice thymectomized three days after birth (NTx mice), with severely reduced numbers of naturally arising Foxp3+ regulatory T cells in periphery, developed inflammation of the liver. However, PD-1−/− BALB/c mice with neonatal thymectomy (NTx-PD-1−/− mice) develop fatal AIH characterized by CD4+ and CD8+ T-cell infiltration, with massive lobular necrosis. Because of the massive destruction of the parenchyma of the liver, these mice start to die as early as two weeks of age, with most dying by four weeks. Notably, the hepatitis in NTx-PD-1−/− mice was characterized by hyper-gammaglobulinemia and huge production of anti-nuclear antibody, both diagnostic hallmarks of AIH patients [18], [19].

In this mouse model, both CD4+ and CD8+ T cells are indispensable for the development of fatal AIH [18], [19]. CD8+ T cells are crucially involved in the progression to fatal hepatic damage, whereas CD4+ T cells are responsible for inducing fatal AIH. Initial activation of CD4+ T cells occurs in the spleen. In the induction phase of AIH, splenic CD4+ T cells were localized in B-cell follicles with huge germinal centers (GCs) and showed the Bcl6+ICOS+IL-21+IL-21R+ follicular helper T (TFH) cell phenotype [19]. IL-21 produced by TFH cells has been shown to drive CD8+ T-cell activation [19]. Splenic TFH cells and activated CD8+ T cells expressed CCR6, and CCL20 expression was elevated in the liver. CCR6-CCL20 axis-dependent migration of splenic T cells is crucial to induce AIH in NTx-PD-1−/− mice [19]. However, it is not known how hepatic CCL20 expression is upregulated in the induction phase of AIH.

AIH-bearing NTx-PD-1−/− mice at three weeks old showed markedly increased levels of TNF-α in the serum [18]. However, it is not clear whether TNF-α is essential in the development of fatal AIH in this mouse model. This study aimed to identify the roles of TNF-α in the development of fatal AIH. We found that serum levels of TNF-α were markedly elevated from the induction phase and that administration of anti-TNF-α prevented induction of AIH, but treatment by anti-TNF-α after the induction did not significantly suppress the progression of fatal hepatic inflammation. Administering anti-TNF-α did not prevent splenic T cell activation in the induction phase of AIH, but suppressed CCL20 expression in the hepatocytes. In contrast, administering anti-CCL20 suppressed fatal AIH but not elevated serum levels of TNF-α. In addition, we found that stimulation by recombinant TNF-α (rTNF-α) upregulated CCL20 expression in hepatocytes in vivo and ex vivo. These findings suggest that TNF-α is critically involved in the induction of fatal AIH in mice through the upregulation of hepatic CCL20 expression.

Section snippets

Mice

BALB/c mice were purchased from Japan SLC (Shizuoka, Japan), and PD-1 deficient mice on a BALB/c background were generated as described previously [20]. These mice were bred and housed under specific pathogen-free conditions. Thymectomy and splenectomy of the mice three days after birth were performed as described previously [18], [19]. All mouse protocols were approved by the Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University.

In vivo neutralization of cytokines and depletion of T cell subsets, and injection of cytokines

NTx-PD-1−/− mice were injected

Production of TNF-α increases in the induction phase of AIH, and neutralizing TNF-α suppresses the development of AIH in NTx-PD-1−/− mice

Previously, we reported that in three-week-old NTx-PD-1−/− mice with severe AIH, serum levels of TNF-α significantly increased [18]. In this study, we performed a time-course study on serum levels of TNF-α from one to three weeks of age. In 1.5-week-old NTx-PD-1−/− mice, the serum level of TNF-α was significantly higher than in PD-1−/− mice (Fig. 1A). The elevated serum level of TNF-α further increased in two-week-old NTx-PD-1−/− mice, at a level similar to NTx-PD-1−/− mice with severe AIH at

Discussion

In the present study, we examined the roles of TNF-α in the development of AIH in mice. We found that not only mice with severe AIH, but also NTx-PD-1−/− mice in the induction phase, showed elevated serum levels of TNF-α. In vivo injection of rTNF-α induced upregulated expression of CCL20 in hepatocytes. Administration of anti-TNF-α suppressed CCL20 expression in the hepatocytes and the infiltration of splenic CD4+ and CD8+ T cells into the liver in the induction phase, preventing the

Acknowledgments

We thank Dr. Dovie Wylie for assistance in preparation of the manuscript; Ms. Chigusa Tanaka for excellent technical assistance; Drs. Tasuku Honjo, Shuh Narumiya, Nagahiro Minato, Shimon Sakaguchi, Takeshi Watanabe, and Ichiro Aramori for critical discussion and suggestions. Funding: The Center for Innovation in Immunoregulative Technology and Therapeutics is supported in part by the Special Coordination Funds for Promoting Science and Technology of the Japanese Government and in part by

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    Conflict of interest: None of the authors has any financial conflict of interest to disclose in relation to this manuscript.

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