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The Journal of Immunology, 2008, 181, 7221 -7229
Copyright © 2008 by The American Association of Immunologists, Inc.
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CD4+CD25+ Foxp3+ Regulatory T Cells Protect against T Cell-Mediated Fulminant Hepatitis in a TGF-β-Dependent Manner in Mice1

Hua-Xing Wei*, Ya-Hui Chuang{dagger}, Bofeng Li*, Haiming Wei*, Rui Sun*, Yuki Moritoki{dagger}, M. Eric Gershwin{dagger}, Zhe-Xiong Lian2,{dagger} and Zhigang Tian2,*

* Institute of Immunology, Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, China; and {dagger} Division of Rheumatology, Allergy, and Clinical Immunology, University of California, Davis, CA 95616


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Regulatory T cells (Tregs), which are characterized by expression of CD4, CD25, and Foxp3, play a crucial role in the control of immune responses to both self and non-self Ags. To date, there are only limited data on their role in physiological and pathological hepatic immune responses. In this study, we examined the role of hepatic Tregs in immune-mediated liver injury by using the murine Con A-induced hepatitis model. Con A treatment was associated with an increased number of Foxp3+ Tregs in liver but not in spleen. Moreover, the expression levels of Foxp3, CTLA-4, glucocorticoid-induced TNF receptor, as well as the frequency of CD103 of Tregs were increased after Con A injection, being significantly higher in liver than in spleen. Depleting CD25+ cells aggravated liver injury, whereas adoptively transferring CD25+ cells or Tregs reduced liver injury in Con A-treated recipients. Con A treatment induced elevated serum levels and hepatic mononuclear mRNA expressions of TGF-β, which were reduced by Tregs depletion. In addition, anti-TGF-β mAbs blocked the suppressive function of Tregs from Con A-treated mice in vitro. Finally, TGF-β receptor II dominant-negative mice, whose T cells express a dominant negative form of TGFβRII and therefore cannot respond to TGF-β, had a higher mortality rate and severer liver injury than normal mice injected with the same dose of Con A. These results indicate that CD4+CD25+ Tregs play an important role in limiting the liver injury in Con A-induced hepatitis via a TGF-β-dependent mechanism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Regulatory T cells (Tregs)3 play a central role in regulating tolerance and immunity by preventing the activation and expansion of autoreactive T cells that lead to autoimmunity as well as by limiting immune responses in allergic diseases, infections, transplantation, graft-vs-host disease, and cancer (1, 2, 3). Several types of Tregs have been described, including Th3 or Tr1 cells acting primarily through the production or regulatory cytokines such as IL-10 and TGF-β. In recent years, Tregs expressing the Foxp3 transcription factor has become recognized as key regulators of immune homeostasis. These Tregs represent a unique lineage of CD4 T cells characterized by a constitutively high expression of CD25 (CD4+CD25+ Tregs), in which Foxp3 has been demonstrated as critically important for both their development and function (4, 5). Foxp3 deficiency results in loss of Tregs and massive multiorgan autoimmunity in scurfy mice and immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome patients (4, 6, 7, 8). Inducible depletion of Foxp3+ Tregs in newborn mice produces a scurfy-like syndrome with splenomegaly, lymphadenopathy, insulitis, and severe skin inflammation (9). Decreasing Foxp3 expression on a per cell basis is sufficient for the induction of autoimmunity as a result of Treg dysfunction (10), indicating that Foxp3 regulates Treg development and function in a dose-dependent, non-binary manner. Other key Foxp3+ Treg associated molecules include CTLA-4, glucocorticoid-induced TNF receptor (GITR), and CD103, the {alpha}E subunit of {alpha}Eβ7, which may identify phenotypically and possibly functionally heterogeneous Treg subpopulations.

Although there is extensive evidence that Tregs are crucial regulators of immune responses to self and non-self Ags (1, 11), the role of Tregs in liver disease is unclear. As has been observed in other autoimmune diseases (1), patients with autoimmune liver diseases, such as autoimmune hepatitis (AIH) (12, 13, 14) or primary biliary cirrhosis (PBC) have been found to exhibit fewer peripheral CD4+CD25+ Tregs than healthy controls (15). These findings suggest that Treg deficiency plays a role in the loss of tolerance in autoimmune liver diseases. Interestingly, healthy daughters and sisters of PBC patients also demonstrate a reduced peripheral Treg frequency, implicating genetic factors in the etiology of PBC. In AIH, the frequency and function of peripheral CD4+CD25+ Tregs was found to correlate inversely with disease activity (12). Tregs were not detected in liver biopsy samples from healthy controls, but were present in liver specimens of patients with various liver diseases, the levels being lowest in PBC liver, intermediate in AIH liver, and highest in liver samples from patients with chronic hepatitis C (15). The detection of high levels of Tregs in liver of patients with chronic hepatitis C is consistent with the results of several other studies in patients with hepatitis B and C. Such studies indicate that persistent HBV and HCV infection correlates with increased levels of Tregs compared with healthy controls or patients with resolved infection and is associated with the ability of these Tregs to suppress hepatitis B and C virus-specific T cell responses (16, 17, 18). Another example of the detrimental consequences of strong Treg activity is the suppression of tumor-specific T cell responses, which can be overcome by the elimination or inactivation of Tregs (1). CD4+CD25+ Tregs are also increased in the blood and peri-tumor regions of patients with hepatocellular carcinoma (HCC) as well as several other cancer types (19, 20, 21). Furthermore, CD4+CD25+ Tregs from HCC patients exhibit significantly greater expressions of TGF-β1 (20). This suggests that tumor-specific T cells in HCC are suppressed on-site by Tregs via immunosuppressive cytokines such as TGF-β1.

In this study, we examined the role of hepatic CD4+CD25+ Tregs in liver injury by using the Con A-induced hepatitis model. Con A-induced liver damage in the mouse is a well-characterized model of immune-mediated liver disease and has been used extensively to elucidate various aspects of human T cell-mediated liver diseases, such as AIH and viral hepatitis (22, 23, 24). We examined the numbers and phenotypic features of Tregs in the livers and spleens of Con A-injected mice and investigated the suppressive function of Tregs by systemically depleting or adoptively transferring hepatic CD25+ cells from mice that had been treated with Con A 24 h previously. In addition, we used in vitro cell coculture experiments to investigate the role of TGF-β in the suppressive function of Tregs from Con A-treated mice. To specifically define the role of TGF-β in Treg suppression of liver disease, we analyzed the effect of Con A injection in TGF-β receptor II dominant-negative (dnTGFβRII) mice. We report herein that CD4+CD25+ Tregs are important in the amelioration of liver injury via a TGF-β-dependent manner.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

C57BL/6 (B6) mice were obtained from Shanghai Laboratory Animal Center, Chinese Academy of Science and maintained under specific pathogen-free conditions. Animal-handling protocols and experimental procedures conform to the guidelines outlined in the Guide for the Care and Use of Laboratory Animals. dnTGFβRII mice were originally developed by R. A. Flavell (25) and were bred onto a B6 background (The Jackson Laboratory) (26) at the University of California animal facility. Foxp3gfp mice containing the Foxp3gfp allele that encodes a GFP-Foxp3 fusion protein were provided by Dr. A.Y. Rudensky (University of Washington, WA) (5) and bred and maintained at the University of California animal facility. All experiments used 6- to 8 wk-old male mice.

Con A-induced hepatitis

A stock solution of Con A (type IV, Sigma-Aldrich) was diluted in pyrogen-free PBS and stored at –20°C. B6 and Foxp3gfp mice were i.v. injected with Con A at a dose of 15 µg per gram of body weight (bw). dnTGFβRII mice were injected with 5 µg of Con A per gram of bw. Control mice were injected with the same volume of pyrogen-free PBS.

Isolation of hepatic and splenic mononuclear cells (MNCs)

Livers and spleens were harvested at the indicated time points and pressed through a 200-gauge stainless steel mesh and suspended in PBS. For hepatic MNCs isolation, after washing once with PBS, the cells were resuspended in 40% Percoll. The cell suspension was overlaid gently on top of 70% Percoll and centrifuged at 2400 rpm for 30 min at room temperature. Hepatic MNCs were obtained from the interphase and washed twice with PBS. Erythrocytes were removed using lysis solution.

Purification of CD25+ hepatic MNCs and hepatic Tregs (CD4+GFP-Foxp3+)

To isolated CD25+ hepatic MNCs, total hepatic MNCs from mice that had been treated with Con A 24 h previously were labeled with FITC-conjugated anti-CD25 mAb (eBioscience) and then incubated with anti-FITC microbeads (Miltenyi Biotec). CD25+ cells were enriched by positive MACS according to the manufacturer’s protocol. The purity of CD25+ cells was consistently >90%. To purify hepatic Tregs (CD4+GFP-Foxp3+), hepatic MNCs from 24 h-treated Foxp3gfp mice were labeled with PE/Cy5-conjugated anti-CD4 mAb (eBioscience) and then sorted by FACSAria Cell Sorter (BD Biosciences).

Isolation of hepatocytes

For the cytotoxicity assay, primary mouse hepatocytes were isolated by collagenase perfusion from naive B6 mice. In brief, mice were anesthetized and livers were perfused with EGTA solution and digested with 0.05% collagenase solution. The suspended hepatocytes were then added on top of 40% Percoll, centrifuged at 1400 rpm for 10 min at 4°C and washed once with DMEM.

Flow cytometry

Cell surface phenotypes were determined by multicolor flow cytometry. Hepatic or splenic MNCs were pre-incubated with anti-CD16/32 (clone 93) to block non-specific FcR{gamma} binding, then stained with mAbs at a dilution that had previously been determined to be optimal. The following Abs were used in this study: anti-CD11c PE/Cy5, anti-CTLA4 PE, anti-GITR PE, anti-CD103 PE (eBioscience); anti-NK1.1 PE/Cy5, anti-CD25 allophycocyanin, anti-CD4 allophycocyanin/Cy7 (Biolegend); and Foxp3 detection kit (Biolegend). Stained cells were assessed on a five-color FACScan flow cytometer (BD Immunocytometry Systems) upgraded by Cytec Development. Acquired data were analyzed with CellQuest Pro (BD Immunocytometry Systems) and FlowJo software (Tree Star).

Adoptive transfer of hepatic CD25+ cells and Tregs (CD4+GFP-Foxp3+)

Hepatic MNCs were obtained as described above from mice that had been treated with Con A 24 h previously. Purified CD25+ hepatic MNCs (5 x 105 cells) or hepatic Tregs (CD4+GFP-Foxp3+, 2 x 105 cells) at a volume of 50 µl were injected into the left lateral lobe of the liver of mice using a 29-gauge needle at a rate of 10 µl/sec. Control mice received untreated hepatic MNCs.

Depletion of CD25+ cells

The anti-mouse-CD25 mAbs were purified from the culture supernatants of hybridoma PC61.5.3 (American Type Culture Collection) by using ammonium sulfate precipitation. To deplete CD25+ cells, mice were i.p. injected with a previously defined optimal dose (0.5 mg) of anti-mouse-CD25 mAbs (clone PC61.5.3) or control Ab 6 h after Con A injection. The cell depletion was confirmed by flow cytometry staining with anti-CD25 mAb clone 7D4 and showed a >90% decrease in the number of the CD25+ cells.

Determination of serum alanine aminotransferase (ALT) activity and cytokine levels

Mice were sacrificed at the indicated time points indicated, 4 and 5, and sera were stored at –20°C for assays of ALT activity and serum cytokine levels. Serum TGF-β1 and IL-10 levels were analyzed using a commercially available ELISA kit (Quantikine, R&D Systems) according to the manufacturers’ instructions. The ALT activities were determined by a commercially available Alanine Aminotransferase Reagent Kit (Rongsheng Biotech) based on methods recommended by the International Federation of Clinical Chemistry.

Isolation of RNA and real-time PCR for cytokine mRNAs

Total RNA was isolated from hepatic MNCs using RNAprep Micro Kit (Tiangen Biotech) according to the manufacturer’s protocol. RNA was than transcribed using SuperScript II Reverse Transcriptase (Invitrogen Life Technologies). Quantitative real-time PCR was conducted according to the manufacturer’s instructions using a SYBR Premix Ex Taq (Takara). Reactions were conducted in reaction tubes (Takara) in a sequence detector (ABI PRISM 7000; Applied Biosystems), beginning with a 10-s hot-start activation of the Taq polymerase at 95°C, followed by 40 cycles of amplification in two steps (denaturation at 95°C for 5 s, followed by a 31-s annealing/extension at 60°C). For analysis, all expression levels of target genes were normalized to the housekeeping gene β-actin ({Delta}Ct). Gene expression values were then calculated based on the {Delta}{Delta}Ct method, using the mean of liver of PBS-treated mice as calibrator to which all other samples were analyzed. Relative hepatic MNCs mRNA expression were determined by 2–{Delta}{Delta}Ct. The primer sequences used were as follows: β-actin sense 5'-TGGAATCCTGTGGCATCCATGAAA-3'; β-actin antisense 5'-TAAAACGCAGCTCAGTAACAGTCCG-3'; TGF-β1 sense 5'-CTCCCACTCCCGTGGCTTCTAG-3'; TGF-β1 anti-sense 5'-GTTCCACATGTTGCTCCACACTGG-3'; IL-10 sense 5'-TCATTCATGGCCTTGTAGACAC-3'; IL-10 anti-sense 5'-AGCTGGACAACATACTGCTAAC-3'.

Cytotoxicity assay

To assay the cytotoxicity of hepatic MNCs against primary mouse hepatocytes, a 4-h aspartate aminotransferase (AST) release assay was used (27). Primary mouse hepatocytes were isolated from naive B6 mice as target cells. Hepatic MNCs (effector cells) were isolated from mice that had been treated with Con A 2 h previously and cocultured with hepatocytes at an E:T ratio of 50:1. CD25+ hepatic MNCs (suppressor cells) were purified and added to the culture mixture at an effector to suppressor ratio of 5:1. Mouse anti-TGF-β neutralizing Ab (clone 1D11, R&D Systems) was added into the culture mixture at a final concentration of 20 µg/ml. After 4 h, the supernatant was harvested and AST activities were measured by using a commercially available Aspartate Aminotransferase Reagent Kit (Rongsheng Biotech). The specific cytotoxicity was calculated as: [(ASTexperimental – ASTspontaneous)/(ASTmaximum – ASTspontaneous)] x 100%.

Histopathology

Livers were fixed in 10% buffered formalin, embedded in paraffin, and tissue sections were then cut into 5 µm slices for routine hematoxylin (DakoCytomation) and eosin (American Master Tech Scientific) staining. The sections were interpreted by a pathologist who was unaware of the protocol, and the observations were recorded by the coded number of the slide.

TUNEL assay

Apoptotic and necrotic damage of the liver was evaluated using an in situ apoptosis detection kit (TACS TdT kit; R&D Systems). In brief, paraffin embedded sections (4 µm) of murine liver were treated with proteinase K after rehydration. After quenching of endogenous peroxidase activity, terminal deoxynucleotidyl transferase labeling was performed. The reaction products were visualized with diaminobenzidine and counsterstained with methyl green.

Statistical analysis

All results are expressed as the mean ± SEM, evaluated with an unpaired two-tailed Student’s t test or a one-way ANOVA, followed by a Tukey’s post hoc test for comparison of more than two groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Phenotype analysis of hepatic and splenic Tregs

To investigate whether Tregs play a role in Con A-induced liver injury, we measured the numbers and characteristics of Tregs in Con A-injected mice. Treatment with Con A induced a significant decrease in the frequency of Tregs (CD4+Foxp3+) in the liver of B6 and Foxp3gfp mice 12 h after injection, followed by a marked increase at the 24-h time point (Fig. 1, A and B). At 48 h after Con A administration, the proportion of hepatic Tregs returned to baseline levels (Fig. 1, A and B). Note that the decreased frequency of Tregs 12 h after Con A injection was due to the massive infiltration of effector T (CD4+CD25+ Foxp3–) cells (Fig. 1B). Interestingly, the increased frequency of Tregs was only seen in this T cell mediated liver injury, but not seen in other immune mediated liver injury by other cells such as NK cell (induced by poly I:C injection; data not shown; Ref. 28) or Kupffer cells (induced by LPS/D-GalN injection; data not shown; Ref. 29). The absolute number of Tregs in liver significantly increased at all time points after Con A injection (Fig. 1C). In the spleens of Con A-treated mice, the frequency of Tregs also declined initially, but did not exhibit the subsequent marked increase as seen in hepatic Tregs at 24 h. In contrast to the increased number of Tregs in liver, there was no significant change in the number of Tregs in spleen following Con A injection (Fig. 1C). The expression levels of Foxp3, assessed as mean fluorescence intensity, were significantly increased in both hepatic and splenic Tregs, but started to decline earlier in splenic Tregs, resulting in significantly higher hepatic Treg Foxp3 expressions at 24 and 48 h after Con A injection (Fig. 1D).


Figure 1
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  		FIGURE 1. Increased number and expression level of Foxp3 of Tregs in the liver of Con A-injected mice. A, Representative flow cytometry analysis of the percentage and expression level (mean fluorescence intensity or MFI) of Foxp3 in the gated population of CD4+ NK1.1– CD11c– cells. B, Graphical representation of the frequency of Tregs (CD4+Foxp3+) and activated T cells (CD4+CD25+Foxp3–) among CD4+ T cells in liver of Con A-injected mice as measured by flow cytometry. C, The absolute numbers of Tregs in livers and spleens of Con A-injected mice were calculated by multiplying the frequency of Tregs by the absolute numbers of hepatic MNCs and splenocytes, respectively. D, Graphical representation of the expression levels of Foxp3 in hepatic and splenic Tregs as analyzed by intracellular flow cytometry. The levels of expression indicated by MFI for each group were calculated. *, p < 0.05; **, p < 0.01; and ***, p < 0.005 compared with 0 h. ##, p < 0.01 and ###, p < 0.005 liver vs spleen. n = 3–5 per group. These results were confirmed in two independent experiments.

 
All Tregs in the spleen and liver expressed intracellular CTLA-4 and cell-surface GITR (Fig. 2A). In contrast, only 52.8% of hepatic Tregs and 39.1% of splenic Tregs expressed CD103 before Con A treatment. The mean expression level of intracellular CTLA-4 was significantly higher in hepatic Tregs compared with splenic Tregs before Con A treatment, whereas GITR expression levels were similar. Con A induced a significant increase in the expression levels of CTLA-4 in hepatic and splenic Tregs, with the expression in hepatic Tregs remaining markedly higher than that in splenic Tregs at all time points (Fig. 2B). Con A also augmented the expression of surface GITR on hepatic and splenic Tregs, with a markedly more pronounced increase observed in hepatic Tregs compared with splenic Tregs (Fig. 2C). After Con A administration, the percentage of CD103+ Tregs was somewhat increased in the liver, but slightly decreased in the spleen (Fig. 2D). Neither of those changes was statistically significant. As a result, however, the frequency of CD103+ Tregs in liver was significantly higher compared with spleen at all time points after Con A injection. Taken together, these results suggest that Tregs in the liver may exert a more potent function than those in the spleen.


Figure 2
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  		FIGURE 2. The expression of CTLA-4, GITR, and CD103 on hepatic and splenic Tregs (CD4+Foxp3+NK1.1–CD11c–) was analyzed by flow cytometry. A, Representative flow cytometry analysis at baseline and 24 h after Con A treatment. B–D, Graphic representation of the changes over time in the expression level of CTLA-4 (B) and GITR (C), and in the percentage of CD103+ cells (D). The expression levels of surface molecules indicated by the MFI for each group were calculated. **, p < 0.01 and ***, p < 0.005 compared with 0 h. ##, p < 0.01 and ###, p < 0.005 liver vs spleen. n = 3–5 per group. These results were confirmed in two independent experiments.

 
Depletion of CD25+ cells aggravated Con A-induced hepatitis

To define the functional role of Tregs in Con A-induced hepatitis, we depleted CD25+ cells in Con A-injected mice by injecting anti-CD25 mAbs 6 h after Con A administration (Fig. 3A). Treatment with anti-CD25 Abs significantly decreased the absolute number of CD4+Foxp3+ cells in the liver of mice injected with Con A (Fig. 3B). Anti-CD25 Abs injection alone did not cause any liver injury (Fig. 3, C and D). Con A-induced elevations in serum ALT concentrations were significantly more pronounced in mice treated with anti-CD25 Abs compared with those treated with control Abs (Fig. 3C). This difference was most striking 24 h after Con A administration but persisted till the 36- and 48-h time points. Moreover, an increased severity in necrotic liver injury was observed in Con A-injected mice treated with anti-CD25 mAbs compared with those treated with isotype control Abs (Fig. 3D).


Figure 3
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  		FIGURE 3. Depletion of CD25+ cells aggravated Con A-induced liver injury. A, Experimental protocol: Mice were treated with either anti-CD25 or control Abs 6 h after Con A or PBS injection. B, The absolute numbers of CD4+Foxp3+ cells in liver in Con A-injected mice were measured 6 h after Ab treatment. *, p < 0.05 isotype vs anti-CD25 Ab. C, Serum ALT levels were measured 12, 24, 36, and 48 h after Con A injection. *, p < 0.05 compared with isotype-treated mice. D, Representative results of histopathologic examination of liver in anti-CD25 Ab-treated and control mice. x100 and x200 magnification. n = 3–5 per group. Similar results were obtained in three independent experiments.

 
Adoptive transfer of hepatic CD25+ cells or Tregs ameliorated Con A-induced liver injury

To further assess the functional role of Tregs in Con A-induced liver injury, we purified hepatic CD25+ cells and Tregs (CD4+GFP-Foxp3+) from mice that had been treated with Con A 24 h previously and adoptively transferred them into another group of mice before Con A treatment (Fig. 4A). The Con A-induced increase in serum ALT levels was significantly suppressed in recipients of either adoptively transferred hepatic CD25+ cells or Tregs if compared with control mice with untreated hepatic MNCs transfers (Fig. 4B). Moreover, only a mild necrotic liver injury appeared in CD25+ cell- or Treg-transferred mice with Con A injection (Fig. 4C). These results suggest that Con A-induced hepatic CD25+ cells and Tregs have a suppressive function in Con A-induced hepatitis.


Figure 4
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  		FIGURE 4. CD25+ cells and Tregs from Con A-treated mice ameliorated Con A-induced liver injury in recipients. A, Experimental protocol. Mice were adoptively transferred with purified hepatic CD25+ cells or Tregs from Con A-treated donors and injected with Con A immediately after the transfer. B, Serum level of ALT in recipient mice was measured 24 h after Con A injection. *, p < 0.05. C, Representative results of histopathologic examination of liver in control mice (left panel), recipients of CD25+ cells (middle panel) and Tregs (right panel). x100 and x200 magnification. n = 3–5 per group. These results were confirmed in three independent experiments.

 
The suppressive function of hepatic CD4+CD25+ Tregs is mediated by TGF-β

IL-10 and TGF-β have been implicated as mediators of Treg suppressor functions in vitro and in vivo (11, 30, 31, 32). As illustrated in Fig. 5, A and B, treatment with anti-CD25 Abs was associated with a significant reduction in the serum levels and the hepatic MNCs mRNA expression levels of TGF-β1 in Con A-injected mice. In contrast, depletion of CD25+ cells did not reduce serum and hepatic IL-10 levels of Con A-injected mice (Fig. 5, A and B). Hepatic MNCs isolated from mice 2 h after Con A administration exhibited significant cytotoxicity against primary mouse hepatocytes in vitro. The addition of hepatic CD25+ cells isolated from mice 24 h after Con A injection significantly suppressed this cytotoxic activity, and this suppression could be blocked by anti-TGF-β mAbs (Fig. 5C). Together, these results indicate that TGF-β1 play a central role in the function of Tregs in Con A-induced liver injury.


Figure 5
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  		FIGURE 5. TGF-β1 is important in the suppressor function of Tregs in Con A-induced liver injury. A and B, Mice were bled and liver tissues were taken at the time points indicated in figures. Serum levels of TGF-β1 (A, left panel) and IL-10 (A, right panel) in Con A-injected mice treated with either anti-CD25 or control Abs were measured by ELISA. B, Cytokine mRNA expression of hepatic MNCs were measured by quantitative real-time RT-PCR. X-fold induction was calculated referring to mRNA levels of the respective cytokines in 0 h Con A-treated mice. *, p < 0.05 compared with anti-CD25 Ab untreated group. C, Cytotoxicity was measured in the present or absence of anti-TGF-β Ab in the culture mixture. *, p < 0.05. Three independent experiments yielded similar results.

 
Increased liver injury in Con A-injected dnTGFβRII mice

To understand whether TGF-β is required for suppressing liver injury induced by effector T cells, we employed dnTGFβRII mice, whose T cells express a dominant negative form of TGFβRII and therefore cannot respond to TGF-β. A high mortality was observed in dnTGFβRII mice injected with a sublethal dose of Con A (15 µg/g bw) compared with B6 mice (Fig. 6D). Even when the dose of Con A was reduced to 5 µg/g bw, dnTGFβRII mice exhibited more pronounced liver pathology and higher serum ALT levels than B6 mice (Fig. 6, A and C). In addition, apoptosis or necrosis of hepatocytes was observed in dnTGFβRII but not B6 mice by the TUNEL assay (Fig. 6B).


Figure 6
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  		FIGURE 6. Increased liver injury in Con A-injected dnTGFβRII mice. dnTGFβRII mice were i.v. injected with 5 µg/g (A–C) or 15 µg/g (D) body weight of Con A. A, Representative results of histopathologic examination in liver in Con A-injected dnTGFβRII and control mice. B, Representative results of TUNEL assay in liver in Con A-injected dnTGFβRII and control mice. C, Serum ALT levels 24 h after Con A injection. n = 5 per group. **, p < 0.01. These results were confirmed in two independent experiments. D, Survival rate for Con A-injected dnTGFβRII and control mice. n = 9.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we examined the role of hepatic CD4+CD25+ Tregs in immune-mediated liver injury by using the Con A-induced hepatitis model. We observed an increased number of Foxp3+ Tregs in the livers but not in the spleens of mice i.v. injected with Con A. Moreover, the expression levels of Foxp3, CTLA-4, and GITR, as well as the frequency of CD103+ cells were increased in Tregs after Con A injection, with significantly higher levels found in liver than in spleen. Depleting CD25+ cells aggravated liver injury while adoptively transferring hepatic CD25+ cells or Tregs from Con A-treated mice reduced liver pathology. Administration of anti-CD25 mAbs attenuated the Con A-induced elevation in serum levels and hepatic MNC mRNA expression of TGF-β1. In addition, neutralizing anti-TGF-β mAbs blocked the suppressive function of Tregs from Con A treated mice in vitro. Finally, dnTGFβRII mice had a higher mortality rate and exhibited more severe liver injury than normal mice injected with the same dose of Con A. Together, our results provide evidence that CD4+CD25+ Tregs are important in limiting liver injury via a TGF-β-dependent mechanism in the Con A-induced hepatitis. We also examined hepatic Tregs in other immune mediated liver injury. Tregs in NK cell-mediated hepatitis model and Kupffer cell-mediated hepatitis model remained unchanged in frequency. The different behavior of Treg in T cell-mediated hepatitis model and other two immune-mediated models remains to be further study.

There is considerable evidence that Treg suppressor functions require cell-cell contact and that the interaction between CTLA-4 on Tregs and its ligands, CD80 and CD86, on effector T cells plays a central role in this mechanism (33). For example, the administration of anti-CTLA-4 Abs blocks CTLA-4 signaling in Tregs and hence inhibits Treg function in vitro (34) and in vivo (35, 36). In contrast, GITR seems to be involved in the inhibition of Treg-mediated suppression (33). Ligation of GITR inhibits the regulatory activity of Tregs, as illustrated by the administration of agonistic anti-GITR Abs that resulted in organ-specific autoimmune disease in normal mice and provoked potent tumor-specific immunity (37, 38). Approximately 25% of mouse Tregs express CD103, the {alpha}E subunit of {alpha}Eβ7, which interacts with E-cadherin, a molecule expressed on epithelial cells (39). CD103+ Tregs appear to have more potent suppressive effects in vivo, possibly due to their preferential migration to inflamed tissue (40). Other results suggest that CD103 has a role in tissue retention rather than homing (41). Specifically, it was reported that CD103 does not characterize a functionally distinct subset of Tregs, but rather is a molecule that is rapidly induced on Tregs upon their arrival in inflamed tissue (41). In this study, Con A induced the expression of surface Treg markers, including CTLA-4, CD103, and GITR, with induction being consistently higher on hepatic than on splenic Tregs (Fig. 2). Although more studies are required to further understand the effects of the elevated expression levels of these cell surface markers, these results suggest that Tregs in the liver may exert more potent regulatory functions than those from the spleen.

In the current study, the adoptive transfer of hepatic CD25+ cells or hepatic Tregs from Con A-treated mice reduced Con A-induced liver pathology in the recipients compared with animals that receive untreated hepatic MNCs. Similarly, it was recently reported that the adoptive transfer of CD4+CD25+ Tregs obtained from mice that had been treated with Con A 8 days prior resulted in significantly lower Con A-induced plasma ALT activity in recipients compared with recipients of CD4+CD25– cells (42). Tregs from PBS-treated donors were somewhat less effective. Conversely, our results demonstrate that depleting CD25+ cells aggravates Con A-induced liver injury (Fig. 3), providing further indication that CD25+ T cells exert a suppressive function in Con A-induced liver injury. However, CD25 is expressed not only on Tregs but also on activated effector T cells. In fact, activated CD4+ T cells are the main cells recruited to the liver in the Con A-induced hepatitis model (Fig. 1B and Ref. 22). The effects of anti-CD25 mAbs on this cell population remains largely unexplored, but may include Ab-induced apoptosis, complement dependent cytotoxicity, or Ab-dependent cell-mediated cytotoxicity. Indeed, there are data suggesting that anti-CD25 mAbs eliminate or functionally inactivate activated effector T cells (37). Nevertheless, this seems unlikely to have occurred in our experiments since the depletion of activated T cells would be expected to result in less pronounced liver injury, whereas we observed more severe liver pathology in anti-CD25 mAb-treated animals, and this was mediated by massive lymphocytic infiltrates that included effector T cells.

Although not significantly affecting the CD25+ effector T cell population, anti-CD25 Abs treatment considerably decreased the number of CD4+Foxp3+ cells compared with control Abs in livers of Con A-injected, but not of PBS-treated, mice (Fig. 3B). Numerous studies have shown, and our own results confirm (data not shown), that treatment with anti-CD25 mAbs results in the rapid and almost complete elimination of CD25+ cells (42, 43). Recent data, however, indicate that such treatment is not (43) or is only partially effective in depleting Tregs as identified by Foxp3 expression (44, 45). In agreement with the small or even negligible effects reported in some of these studies conducted with naive untreated animals (43, 44), we observed essentially no change in the number of Foxp3+ Tregs in the liver of PBS-treated mice. In marked contrast, we noted a pronounced decrease in the number of hepatic Foxp3+ Tregs in Con A-treated mice. This would seem to suggest that Con A renders hepatic Tregs more susceptible to depletion by anti-CD25 mAb treatment, which may be due to the critical role of IL-2 in the peripheral induction and development of Tregs (46, 47). In fact, internalization or shedding of CD25, the {alpha}-chain of the high-affinity IL-2 receptor, is associated with the functional inactivation of Tregs (43, 44), although this is not an entirely consistent finding (45). This makes it likely that not only Treg depletion but also the inability to induce Foxp3+ Tregs from naive T cells in the absence of IL-2 signaling and perhaps a decrease in Treg function due to anti-CD25 treatment all contributed to the enhanced liver pathology seen in anti-CD25 mAb-treated mice.

In addition to cell contact-dependent mechanisms, the ability of Tregs to secrete immunosuppressive cytokines (IL-10 and TGF-β) or to induce their production in other immune cells has been implicated as a regulatory mechanism in some models (31, 32, 33, 36, 48). Several groups of investigators have used dnTGFβRII mice to demonstrate that the ability of effector T cells to respond to TGF-β is central to the suppressor activity of Tregs in diverse models (49, 50). Consistent with these findings, we observed a higher mortality rate in dnTGFβRII mice injected with Con A (15 µg/g bw) compared with B6 mice (Fig. 6D). More pronounced liver injury and markedly higher serum ALT levels were also noted in dnTGFβRII mice relative to normal B6 mice when both groups were injected with a lower dose of Con A (5 µg/g bw) (Fig. 6). These results suggest that TGF-β is needed for suppressing liver injury induced by effector T cells. Recently, Erhardt et al. (42) obtained somewhat different results when they treated mice with Con A twice, once on day 0 and once 8 days later (42), different from our experiment by dynamic observation of once injection of Con A. In Erhardt et al.’s study, compared with pretreatment with saline, pretreatment with Con A was associated with attenuation of hepatic necrosis and plasma ALT activity upon rechallenge with Con A despite similar levels of lymphocyte infiltration in the liver. The results from mechanistic studies indicate that Tregs were important in reducing liver pathology by producing IL-10 or inducing its secretion in other cell types. In this model, TGF-β levels in plasma and mRNA expressions in livers were not significantly different between animals that had been pretreated with Con A and those that had not. This is in contrast with our finding that depletion of CD25+ cells did not significantly reduce serum and hepatic IL-10 levels of Con A-injected mice (Fig. 5, A and B), but was associated with a significant reduction in the serum levels and hepatic MNC mRNA expression levels of TGF-β1 in Con A-injected mice. This discrepancy is most likely due to the different models used Con A treatment was used only once in our experiments compared with Con A rechallenge 8 days after the primary administration of Con A in the study by Erhardt et al. (42). The time points of cytokine detection were also different from that in the study by Erhardt et al.

Our results clearly indicate that TGF-β plays a central role in mediating the suppressive function of Tregs both in vivo in the Con A-induced hepatitis model and in vitro in the suppression of hepatic MNC-mediated cytotoxicity (Figs. 5 and 6).


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 Funding provided by Natural Science Foundation of China (30721002, 30730084, 30630059, 30671901, and 30570819), Ministry of Science & Technology of China (973 Basic Science Project 2007CB512405, 2007CB512807, 2006CB504300, and 2004CB518807), and National Institutes of Health grants (DK074768, DK39588, N01-DK-9-2310, and R21DK077961). Back

2 Address correspondence and reprint requests to Dr. Zhigang Tian, School of Life Sciences, University of Science and Technology of China, 443 Huangshan Road, Hefei, Anhui, China. E-mail address: tzg{at}ustc.edu.cn; or Dr. Zhe-Xiong Lian, Division of Rheumatology, Allergy and Clinical Immunology, University of California at Davis School of Medicine, 451 East Health Sciences Drive, 6605A, Davis, CA 95616. E-mail address: zxlian{at}ucdavis.edu Back

3 Abbreviations used in this paper: Treg, regulatory T cell; GITR, glucocorticoid-induced tumor necrosis factor receptor; AIH, autoimmune hepatitis; PBC, primary biliary cirrhosis; HCC, hepatocellular carcinoma; dnTGFβRII, TGF-β receptor II dominant-negative; bw, body weight; MNC, mononuclear cell; ALT, alanine aminotransferase; AST, asparate aminotransferase. Back

Received for publication October 24, 2007. Accepted for publication September 12, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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