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 Astellas Pharma Inc. in the Formation of the Innovation Center for Fusion of Advanced Technologies Program. This work is partially supported by Grants-in-Aid for Scientific Research (20390207, 21229009, and 23590973) from the Japan Society for the Promotion of Science, a Health and Labor Sciences Research Grant for Research on Intractable Diseases, and Research on Hepatitis from the Ministry of Health, Labor and Welfare, Japan, Grants-in-Aid for Research by The Kato Memorial Trust for Nambyo Research, and The Waksman Foundation of Japan.
Potential conflict of interest: Nothing to report.
Clinical manifestations of autoimmune hepatitis (AIH) range from mild chronic to acute, sometimes fulminant hepatitis. However, it is unknown how the progression to fatal hepatitis occurs. We developed a mouse model of fatal AIH by inducing a concurrent loss of forkhead box P3+ regulatory T cells and programmed cell death-1 (PD-1)-mediated signaling. In this model, dysregulated follicular helper T cells in the spleen are responsible for the induction, and the C-C chemokine receptor 6/C-C chemokine ligand 20 axis is crucial for the migration of these T cells into the liver. Using this fatal AIH model, we aimed to clarify key molecules triggering fatal AIH progression. During progression, T-bet together with interferon (IFN)-γ and C-X-C chemokine receptor (CXCR)3 were highly expressed in the inflamed liver, suggesting helper T (Th)1-type inflammation. T cells that dominantly expanded in the spleen and the inflamed liver were CXCR3-expressing CD8+ T cells; depletion of these CD8+ T cells suppressed AIH progression. Expression of one CXCR3 ligand, chemokine (C-X-C motif) ligand (CXCL)9, was elevated in the liver. CXCL9-expressing macrophages/Kupffer cells were colocalized with infiltrating T cells, and in vivo administration of anti-CXCL9 suppressed AIH progression. In addition, serum levels of interleukin (IL)-18, but not IL-1β, were elevated during progression, and dendritic cells in the spleen and liver highly produced IL-18. In vivo administration of anti-IL-18R suppressed the increase of splenic CXCR3+ T cells and the progression to fatal AIH. Moreover, tumor necrosis factor alpha, but not IFN-γ, was involved in up-regulating CXCL9 in the liver and for increased serum levels of IL-18. Conclusion: These data suggest that, in our mouse model, fatal progression of AIH is mediated by IL-18-dependent differentiation of T cells into Th1 cells and effector T cells, respectively, and that CXCR3-CXCL9 axis-dependent migration of those T cells is crucial for fatal progression. (Hepatology 2014;60:224–236)
Human autoimmune hepatitis (AIH) typically presents as asymptomatic or mild chronic hepatitis. However, presentation as acute severe hepatitis also occurs, and some of these AIH patients manifest liver failure at initial presentation.[1, 2] Untreated patients with severe AIH rapidly decline, with a mortality rate of up to 50% from 3 to 5 years after diagnosis. Patients progressing to acute liver failure respond poorly to corticosteroid treatment, some of them needing liver transplantation (LT).[4, 5] In addition, approximately 20%-30% of patients undergoing LT for AIH develop features of recurrent disease; in some, recurrent AIH behaves more aggressively, with progression to cirrhosis and graft failure. However, it is unknown how this progression to fatal hepatic damages occurs.
Recently, we developed a mouse model of spontaneous fatal AIH.[7-11] Neither programmed cell death-1 (PD-1)-deficient mice (PD-1−/− mice) nor BALB/c mice thymectomized 3 days after birth (NTx mice) developed any inflammation of the liver. However, in PD-1−/− BALB/c mice with neonatal thymectomy (NTx–PD-1−/− mice), immune dysregulation by a concurrent loss of naturally arising forkhead box P3+ regulatory T cells (Tregs) and PD-1-mediated signaling induced fatal AIH. Massive destruction of the parenchyma of the liver resulted in most mice dying by 4 weeks. Fatal AIH in NTx–PD-1−/− mice was characterized by CD4+ and CD8+ T-cell infiltration with massive lobular necrosis in the liver and by hypergammaglobulinemia and production of antinuclear antibodies (ANAs).[7, 8]
In our mouse model of fatal AIH, we identified induction sites, responsible T-cell subsets, and key molecules for induction of AIH. The spleen is an induction site for fatal AIH, and splenic CD4+ T cells were autonomously differentiated into follicular helper T (TFH) cells in 2-week-old NTx–PD-1−/− mice. TFH cells expressing Bcl6, interleukin (IL)−21, IL-21 receptor, inducible costimulator (ICOS), and C-X-C chemokine receptor (CXCR)5 comprise a newly defined effector T-cell subset that powerfully assists B cells in forming germinal centers (GCs). Indeed, in NTx–PD-1−/− mice, the dysregulated TFH cells promoted hypergammaglobulinemia and ANA production. In addition, these TFH cells in the spleen directly migrated into the liver through the C-C chemokine receptor 6/C-C chemokine ligand 20 (CCR6-CCL20) axis, triggering induction of fatal AIH.
On the other hand, in the progression phase of AIH in 3-week-old NTx–PD-1−/− mice, infiltrated CD4+ and CD8+ T cells in the liver produced large amounts of inflammatory cytokines, such as interferon-gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α).[7, 8] Therefore, dysregulated TFH cells in the induction and helper T (Th)1 cells with effector CD8+ T cells in the progression may play their roles at different time points in development of fatal AIH. In this study, using our mouse model, we examined mechanisms in the progression process to identify key molecules triggering fatal AIH progression. We found that in the progression, CXCR3-expressing Th1 cells and CD8+ effector T cells infiltrated in the liver, with CD8+ effector T cells triggering the fatal destruction of the liver, that hepatic macrophages/Kupffer cells (KCs) producing chemokine (C-X-C motif) ligand (CXCL)9 is critical for migration of these T cells, and that dendritic cell (DC)-derived IL-18 is critical for differentiation of Th1 cells and CD8+ effector T cells. These data suggest that in this mouse model of AIH, IL-18 and the CXCR3/CXCL9 axis are critical for T-cell differentiation and migration in fatal progression of AIH.
Materials and Methods
BALB/c mice were purchased from Japan SLC (Shizuoka, Japan), and PD-1−/− on a BALB/c background were generated as described previously. These mice were bred and housed under specific pathogen-free conditions. Thymectomies were performed as described.[7-11] All mouse protocols were approved by the Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University (Kyoto, Japan).
All other protocols for histological and immunohistological (IHC) analysis, real-time quantitative reverse-transcription polymerase chain reactions (qRT-PCR), flow cytometry (FCM) analysis and isolation of single cells, administration of antibodies (Abs) in vivo, enzyme-linked immunosorbent assay (ELISA), in vivo injection of cytokines, DC coculture, and statistical analysis are detailed in the Supporting Materials and Methods.
T-bet, IFN-γ, and CXCR3 Are Highly Up-Regulated in Inflamed Livers of 3-Week-Old NTx-PD-1−/− Mice
In our mouse model, AIH induction was started as early as 2 weeks of age by dysregulated TFH cells in the spleen.[7, 8] Livers in 2-week-old NTx–PD-1−/− mice showed mononuclear cell (MC) infiltrations, predominantly in the portal area, as previously described (Fig. 1A). Within 7 days of induction, these MC infiltrations rapidly progressed and were followed by massive destruction of the parenchyma of the liver (Fig. 1A). To investigate whether cytokines contributed to the severely inflamed livers of these 3-week-old NTx–PD-1−/− mice, we performed real-time qRT-PCR analysis to measure the expression levels of messenger RNA (mRNA) encoding T-cell lineage-specific transcription factors and various related cytokines. In contrast to expression of Th2 or Th17-related molecules, expression of Th1 lineage-specific transcription factor T-bet, together with IFN-γ and TNF-α, were up-regulated in inflamed liver tissues of these mice (Fig. 1B). These data suggest that inflammatory cytokines related to Th1-type inflammation may be involved in fatal progression of AIH. Notably, we found that in the inflamed livers of these mice, mRNA expressions of CXCR3 were highly up-regulated along with Th1-related molecules (Fig. 1C). Although AIH induction was mediated by dysregulated TFH cells in the spleen, in the progression phase of AIH, Th1-type responses were predominant.
T Cells Dominantly Expanded in the Inflamed Liver Were CXCR3-Expressing CD8+ T Cells
Next, we monitored T-cell numbers of the liver, spleen, and mesenteric lymph nodes (MLNs) in NTx–PD-1−/− mice from 1 to 3 weeks of age (Fig. 1D). In the AIH progression phase in 3-week-old mice, we found that CD8+ T cells, and, to a lesser extent, CD4+ T cells, extensively increased in the liver, as described previously. Notably, the predominant increase of CD8+ T cells at 3 weeks was observed only in the liver, but not in the spleen or MLNs (Fig. 1D), implying that CD8+ T cells had accumulated in the severely inflamed liver. In addition, we analyzed splenic and hepatic CD8+ T-cell expression of the chemokine receptors, CCR6, CCR9, and CXCR3, by FCM. As with CD4+ T cells in the spleen and liver, splenic and hepatic CD8+ T cells mainly expressed CXCR3 in 3-week-old NTx–PD-1−/− mice (Fig. 1E).
CD8+ T Cells During Progression of AIH Were Indispensable for Fatal Destruction of the Liver
In a previous study, we showed that in the induction of fatal AIH, CD4+ T cells are indispensable for recruiting CD8+ T cells in the liver and that CD8+ T cells may be major effector T cells, fatally destroying the liver in AIH progression. To examine whether depletion of CD8+ T cells in the progression is sufficient to suppress fatal liver destruction, AIH-developed NTx–PD-1−/− mice were injected intraperitoneally (IP) at 14 days after NTx and then once a week with anti-CD8 monoclonal Abs (mAbs) in vivo (Fig. 2A). After two injections of anti-CD8, the number of CD8+ T cells in the spleen was greatly reduced (Fig. 2B). Although depleting CD8+ T cells did not completely suppress hepatic infiltrations of MCs, infiltration of CD4+ and CD8+ T cells was diminished, and fatal progression of AIH was suppressed by the treatment (Fig. 2C-E). These data suggest that CXCR3-expressing CD8+ T cells extensively infiltrating the liver are indispensable for fatal progression.
Production of a CXCR3 Ligand, CXCL9, Was Elevated in Fatal Progression of AIH
CXCR3-expressing T cells can be guided by three ligands—CXCL9/monokine induced by IFN-γ (MIG), CXCL10/IFN-inducible protein 10 (IP10), and CXCL11/IFN-inducible T cell alpha chemoattractant (I-TAC)—and expression of these CXCR3 ligands in the inflamed tissues determines inflamed-tissue–specific infiltration of CXCR3-expressing T cells in various immunoinflammatory settings, including autoimmune diseases.[14-17] We performed real-time qRT-PCR analysis to measure expression levels of mRNA encoding these three CXCR3 ligands. In contrast to noninflamed livers in control mice, severely inflamed livers of 3-week-old NTx−PD-1−/− mice showed markedly elevated gene expression of CXCL9, but not of CXCL10 and CXCL11 (Fig. 3A). In contrast to inflamed livers, no organs, except those with inflamed gastric tissues, showed a significantly increased level of mRNA expression of CXCL9 (Fig. 3B).
In addition, we confirmed elevated protein expression of CXCL9 only in the inflamed liver, but not the stomach, by IHC (Fig. 3C and Supporting Fig. 1). Furthermore, when we looked at serum concentrations of CXCL9 and CXCL10 at 1-4 weeks of age, serum level of CXCL9, but not CXCL10, at 3-4 weeks of age, was significantly higher than controls (Fig. 3D). These data suggest that CXCL9 plays a key role in progression of AIH.
In Fatal Progression of AIH, the CXCR3-CXCL9 Axis Was Crucial for T-Cell Migration Into the Liver
To determine whether the axis formed by CXCR3 and its ligands contributes to T-cell migration leading to fatal progression of AIH, NTx–PD-1−/− mice were injected IP at 1 day after NTx and then once a week with anti-CXCL9 and/or anti-CXCL10 mAbs in vivo. After four injections, in contrast to anti-CXCL10 injections, anti-CXCL9 injections induced a significantly higher survival rate (Fig. 4A,B). Administering anti-CXCL9 and a combination with anti-CXCL9 and anti-CXCL10, but not anti-CXCL10 alone, greatly reduced infiltration of CD4+ and CD8+ T cells into the liver and liver destruction at 4 weeks (Fig. 4C). These data suggest that in the progression phase of fatal AIH, the CXCR3-CXCL9 axis is crucial for migration of Th1 cells and effector CD8+ T cells into the liver.
The Main Cellular Source of CXCL9 Was Hepatic Macrophages/KCs in Progression of AIH
Next, we examined which cell types express CXCL9 in the inflamed liver by IHC. We found that the majority of CXCL9-expressing cells in the inflamed liver were F4/80 antigen-positive macrophages/KCs and that CD4+ and CD8+ T cells were colocalized with CXCL9-expressing cells in the inflamed liver (Fig. 5A).
In AIH progression, mRNA expression of IFN-γ and TNF-α in the inflamed liver as well as serum levels of these cytokines were markedly elevated,[7-10] and IFN-γ mediated the induction of all three CXCR3 ligands (CXCL9, CXCL10, and CXCL11).[14, 15] When we injected IP with 10 mg/kg of recombinant (r) IFN-γ and TNF-α in 4-week-old PD-1−/− mice, after 2 hours, IFN-γ and TNF-α significantly up-regulated mRNA expression of both CXCL9 and CXCL10 in the liver. Interestingly, we found sustained CXCL9 up-regulation by TNF-α (Fig. 5B). Indeed, neutralization of TNF-α, but not IFN-γ, suppressed hepatic CXCL9-expression in 4-week-old NTx–PD-1−/− mice (Fig. 5C).
In NTx–PD-1−/− mice, TNF-α is essential in the induction of AIH by up-regulating hepatic CCL20 expression, allowing TNF-α-producing activated T cells to migrate from the spleen into the liver. In AIH progression, IHC for TNF-α revealed TNF-α production in several infiltrating cell types (Fig. 5D, left panels), suggesting that TNF-α-dependent up-regulation of CXCL9 expression may be induced by hepatic macrophages/KCs in autocrine fashion and/or by activated T cells in paracrine fashion. However, after migration of TNF-α-producing activated T cells into the liver, neutralizing serum levels of TNF-α could not suppress CXCL9 expression in the liver and serum levels of CXCL9 (Fig. 5D, right panels, and 5E). These data suggest that TNF-α secretion in autocrine and/or paracrine fashion may induce uncontrollable CXCL9 expression in progression of AIH, resulting in unsuccessful anti-TNF-α monotherapy, as previously described.
Serum Levels of IL-18 Were Elevated in AIH Progression, and In Vivo Administration of Blocking Abs for IL-18R Signaling Suppressed Development of Fatal AIH
IL-12 is decisive in the development of Th1 subsets. A recent study showed that IL-12 can trigger naïve T cells to transitionally differentiate into T cells with features of TFH and Th1 cells.[18, 19] However, neutralizaing IL-12p40 did not suppress hepatic inflammation, as described previously (Supporting Fig. 2). In addition, although IFN-γ has been shown to be essential for IL-12-induced Th1 differentiation, neutralizing it did not suppress development of AIH. These data suggest that IL-12 is not exclusively involved in differentiation into T cells with features of Th1 cells in progression of fatal AIH.
Serum levels of IL-18 are increased in patients with AIH and fatal hepatitis.[21, 22] IL-18 is critical for liver injury in mice sequentially treated with Propionibacterium acnes and lipopolysaccharide, and for acute hepatic injury induced by concanavalin A.[23, 24] When we looked at serum levels of IL-18 at 1-3 weeks of age, those of IL-18, but not IL-1β, were elevated, and IL-18 elevation gradually increased through the progression of AIH (Fig. 6A,B). IL-18 signals through the IL-18 receptor complex (IL-18R), and IL-18R contains the heterodimer IL-18Rα and IL-18Rβ subunits. The IL-18Rα subunit is responsible for extracellular binding of IL-18, whereas the IL-18Rβ subunit is nonbinding, but confers high affinity binding for the ligand, and is responsible for biological signals.[25, 26] Therefore, to examine the roles of IL-18 in AIH development, NTx–PD-1−/− mice at 1 day after thymectomy were injected with IL-18Rβ mAb, which can neutralize the IL-18-mediating biological function in IL-18R-expressing cells. Administering anti-IL-18Rβ, but not anti-IL-1β, suppressed MC infiltration, including CD4+ and CD8+ T cells, in the liver (Fig. 6C,D), resulting in decreased serum concentrations of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and a significantly increased survival rate at 4 weeks of age (Fig. 6E,F). These data indicate that IL-18-mediated signaling is critical for development of fatal AIH in NTx–PD-1−/− mice.
IL-18 Is Mainly Produced by DCs in Spleen and Liver of NTx–PD-1−/− Mice
Next, we investigated how IL-18 mediates fatal AIH progression in NTx–PD-1−/− mice. We isolated MCs from liver and spleen of 2.5-week-old NTx–PD-1−/− mice and purified them to CD3+CD4+ T cells, CD3+CD8+ T cells, B220+ B cells, CD11b+CD11c− macrophages, CD11c+ DCs, and CD3−DX5+ natural killer (NK) cells, then measured mRNA expression of IL-18. We found that isolated splenic and hepatic DCs increased IL-18 mRNA expression, together with up-regulated expression of NACHT, LRR, and pyrin domain-containing protein 3 (NALP3) and, to a lesser extent, IL-1β (Fig. 7A,B). In contrast, when we cultured isolated splenic DCs, IL-18, but not IL-1β, was secreted from DCs from NTx–PD-1−/− mice, but not from PD-1−/− mice (Fig. 7C and data not shown). These data suggest that in NTx–PD-1−/− mice, DCs noncanonically secrete IL-18 by activating inflammasome and promoting further differentiation of CD4+ T cells into Th1 cells and CD8+ T cells into effector T cells, respectively.
DCs as Well as CD4+ and CD8+ T Cells in Spleen and Liver Expressed IL-18R in NTx–PD-1−/− Mice
To evaluate whether DCs secreting IL-18 directly or indirectly modulate differentiation of T cells in NTx–PD-1−/− mice, we next examined mRNA expression of IL-18Rα on CD4+ and CD8+ T cells in the spleen and liver. We isolated these cells in spleen and liver of 2.5-week-old NTx–PD-1−/− mice and measured mRNA expression of IL-18Rα. We found that isolated splenic and hepatic CD4+ and CD8+ T cells increased IL-18Rα mRNA expression, suggesting that IL-18 can directly affect differentiation of these cells (Fig. 7D). Interestingly, isolated DCs in spleen and liver of 2.5-week-old mice expressed up-regulated expression of IL-18Rα mRNA (Fig. 7E). In addition, 4-week-old NTx–PD-1−/− mice injected with anti–IL-18Rβ mAb showed decreased serum levels of IL-18 (Fig. 7F), suggesting that IL-18 may act as an autocrine for differentiation and/or function of proinflammatory IL-18R-expressing DCs. In AIH progression, mRNA expression of IFN-γ and TNF-α in the inflamed liver as well as serum levels of these cytokines were markedly elevated,[7-10] so TNF-α could be involved in the maturation of DCs. Indeed, neutralization of TNF-α, but not IFN-γ, reduced serum levels of IL-18 (Fig. 7F), implying that TNF-α is also directly/indirectly involved in differentiation and/or function of proinflammatory IL-18R-expressing DCs.
Neutralization of IL-18R Signaling Altered Splenic T-Cell Function and Ab Production in NTx–PD-1−/− Mice
We found that DCs and T cells—not only in inflamed liver, but also in the spleen—expressed IL18Rα in AIH progression (Fig. 7D,E). In addition, CD4+ and CD8+ T cells in the spleen predominantly expressed CXCR3 (Fig. 1E). We next examined whether IL-18 is involved in differentiation of splenic T cells in NTx–PD-1−/− mice. We found that injecting anti-IL-18Rβ significantly reduced the number of CXCR3+ cells in CD4+ T cells as well as in CD8+ T cells of the spleen in 2.5-week-old NTx–PD-1−/− mice (Fig. 8A). In addition, we found that neutralizing IL-18-mediated signaling suppressed expression of T-bet, IFN-γ, TNF-α, and IL-18Rα and up-regulated expression of GATA3 in splenic CD4+ T cells (Fig. 8B). Moreover, although production of total immunoglobulin (Ig) and ANA increased in NTx–PD-1−/− mice, injecting anti-IL-18Rβ reduced total Ig and ANA in the Th1-dependent IgG2a subclass (Supporting Fig. 3). In this mouse model, splenic CD4+ T cells showing the TFH cell phenotype were localized in B-cell follicles with huge GCs. Although injections of anti-IL-12p40 did not significantly reduce the size of GCs in the spleen at 4 weeks, injecting anti-IL-18Rβ mAb induced enlargement of peanut agglutinin+ GC in B220+ follicles (Supporting Fig. 4A,B). Taken together, these data suggest that DC-derived IL-18 is involved in differentiation of CD4+T cells into Th1 cells and CD8+ T cells into effector T cells, respectively, in spleen of NTx–PD-1−/− mice.
In this study, using our fatal AIH model, we examined molecules key to the triggering of fatal progression of AIH. We found that in the progression, CXCR3 expressing Th1 cells and CD8+ effector T cells infiltrated the liver, with CD8+ effector T cells triggering the fatal destruction of the liver, that hepatic macrophages/KCs producing CXCL9 is critical for migration of these T cells, and that DC-derived IL-18 is critical for differentiation of Th1 cells and CD8+ effector T cells (Fig. 8C).
We previously reported that in the induction phase of AIH in 2-week-old NTx−PD-1−/− mice, IL-21-producing splenic TFH cells directly migrated into the liver through the CCR6-CCL20 axis, triggering AIH. In contrast, we showed here that in severely inflamed livers in 3-week-old NTx−PD-1−/− mice, DC-derived IL-18 mediates differentiation of Th1 cells and CD8+ effector T cells, and the CXCR3-CXCL9 axis triggers the migration of these T cells, resulting in fatal AIH progression. Therefore, in the development of fatal AIH in our model, different types of T cells are critically involved at different time points in the induction and fatal progression of AIH. This involvement has also been reported in experimental autoimmune encephalomyelitis (EAE), a CD4+ T-cell-mediated disease of the central nervous system (CNS). In EAE, Th17 cells migrate through the CCR6-CCL20 axis, triggering inflammation in the induction phase, whereas Th1 cells are mainly involved in inflamed lesions in the CNS during active progression. In addition, a recent study reported that TFH-like cells were transiently generated during IL-12-mediating Th1 cell differentiation. In mice infected with Toxoplasma gondii, an obligate intracellular parasite, TFH-like cells were generated 7 days after infection, the proportion of TFH-like cells declined, and IFN-γ-producing Th1 cells increased at day 15.
In this study, we showed that DC-derived IL-18 is critical for differentiation of Th1 cells and CD8+ effector T cells in AIH progression. IL-18 is known to be produced by various types of immune cells and epithelial cells.[25, 26] In humans, IL-18 produced by DCs promotes Th1 induction. IL-18 stimulates Th1-mediated immune responses and activates Th1 cells, which highly express functional IL-18 receptor, producing large amounts of IFN-γ.[25, 26] In addition, in an atopic dermatitis mouse model, IL-18 could induce differentiation of Th1-like cells that expressed IFN-γ and CXCR3. In humans, IL-18 has been shown to be involved in disease processes associated with excessive Th1 responses in several inflammatory diseases, including autoimmune diseases.[30-32] Patients with acute hepatitis, chronic liver disease, fulminant hepatitis, primary biliary cirrhosis, or AIH all show elevated serum levels of IL-18,[21, 22] which correlates with disease severity.[33, 34]
We found that splenic and hepatic DCs increased IL-18 mRNA expression, together with up-regulated expression of NALP3 and, to a lesser extent, IL-1β. However, DCs only secreted IL-18 and induced elevation of serum levels of IL-18. Indeed, administering anti-IL-18Rβ, but not anti-IL-1β, suppressed fatal AIH. After inflammasome activation by NALP3 occurs in the cells, inactive pro-caspase-1 is activated into active caspase-1. Subsequent to cleavage by active caspase-1, mature IL-18 as wells as IL-1β can be secreted from cells. These canonical IL-1β and IL-18 secretions by inflammasome activation are involved in acetaminophen-induced liver injury. However, several recent studies suggest that secretion of IL-18, but not IL-1β, by activation of inflammasome and caspase-1 can be orchestrated by several distinct regulatory mechanisms.[37-39] Thus, in NTx–PD-1−/− mice, distinct licensing of IL-1β and IL-18 secretion may be involved in the noncanonical secretion of IL-18 by activation of inflammasome.
Because TNF-α can directly induce maturation of DCs, TNF-α and IL-18 may directly induce inflammasome up-regulation and skew toward IL-18 production through repression of IL-1β transcript, but up-regulation of IL-18 transcript. On the other hand, TNF-α directly and indirectly induces cell death of hepatocytes and free DNA released from apoptotic hepatocytes can activate Toll-like receptor 9, triggering a signaling cascade to induce pro-IL-1β and pro-IL-18. Therefore, TNF-α may induce apoptosis of hepatocytes, triggering canonical IL-18 production initially. However, IL-18 may act as an autocrine for skewing prolonged IL-18 secretion in DCs.
Although first described as IFN-γ-inducing factor, IL-18 may not make a major contribution to elevated serum levels of IFN-γ in AIH progression. In contrast to IL-18, serum levels of IFN-γ reached the maximal level at 1 week of age before AIH development; the elevated serum level of IFN-γ gradually decreased during AIH progression. Indeed, IFN-γ was dispensable for up-regulating CXCL9 in the liver. Neutralizing IFN-γ did not prevent development of AIH and induced increased T-cell proliferation in the spleen and liver, resulting in exacerbated T-cell infiltration in AIH. So, although IFN-γ generally acts as a critical proinflammatory mediator, it exerts regulatory functions to limit tissue damage associated with inflammation of AIH in progression.
We showed here that migration of exclusively CXCR3-expressing T cells was triggered by hepatic macrophages/KCs producing one CXCR3 ligand, CXCL9. Although CXCL9, CXCL10, and CXCL11 can bind to the common receptor, CXCR3, differences have been reported in the kinetics and the tissue/cell-type expression patterns of these three chemokine genes and their proteins during infection or inflammatory responses.[41-44] Studies using CXCL9- or CXCL10-deficient mice have shown the nonredundant function of these chemokines in various immunoinflammatory settings, including a hepatitis B virus transgenic mouse model and a liver injury model.[41-44]
In this study, we showed that CXCL9-expressing cells are macrophages/KCs in AIH progression. Although recombinant (r)IFN-γ and rTNF-α up-regulated hepatic CXCL9 expression, anti-IFN-γ did not suppress hepatic CXCL9 up-regulation. In NTx–PD-1−/− mice, cell types responsible for secreting CXCR3 ligands in various organs may exhibit a refractory response to constitutively elevated serum IFN-γ. In addition, TNF-α secreted in autocrine and in paracrine fashion by activated T cells may induce uncontrollable CXCL9 expression in AIH progression. Therefore, anti-TNF-α monotherapy may not significantly prevent fatal AIH in mice.
In conclusion, we have identified the pivotal role of the IL-18 and the CXCR3-CXCL9 axis in fatal progression of AIH, implying that blocking these systems may have clinical potential for protecting against fatal progression of this disease.
The authors thank Dr. Taku Okazaki and Tasuku Honjo for providing PD-1-deficient mice, Dr. Dovie Wylie for assistance in preparation of the manuscript, Ms. Chigusa Tanaka for her excellent technical assistance, and Drs. Shuh Narumiya, Nagahiro Minato, Shimon Sakaguchi, Takeshi Watanabe, and Ichiro Aramori for their critical discussion and suggestions.