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Keywords:

  • CD8+ T cells;
  • dnTGF-βRII mice;
  • glucosylceramide;
  • liver inflammation;
  • primary biliary cirrhosis

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

We have demonstrated spontaneous development of autoimmune cholangitis, similar to human primary biliary cirrhosis, in mice expressing a dominant negative form of the transforming growth factor-β receptor (dnTGF-βRII) restricted to T cells. The autoimmune cholangitis appears to be mediated by autoreactive CD8+ T lymphocytes that home to the portal tracts and biliary system. Because the liver pathology is primarily secondary to CD8+ T cells, we have determined herein whether administration of β-glucosylceramide (GC), a naturally occurring plant glycosphingolipid, alters the natural history of disease in this model. We chose GC because previous work has demonstrated its ability to alter CD8+ T cell responses and to down-regulate tissue inflammation. Accordingly, dnTGF-βRII mice were treated with either GC or control for a period of 18 weeks beginning at 6 weeks of age. Importantly, in mice that received GC, there was a significant decrease in the frequency and absolute number of autoreactive liver-infiltrating CD8+ T cells, accompanied by a significant decrease in activated CD44high CD8+ T cell populations. Further, there was a significant reduction in portal inflammation in GC-treated mice. Interestingly, there were no changes in anti-mitochondrial antibodies, CD4+ T cells, CD19+ B cells or natural killer (NK) T cell populations, indicating further that the beneficial effects of GC on liver inflammation were targeted specifically to liver-infiltrating CD8+ T cells. These data suggest that further work on GC in models of CD8+ T-mediated inflammation are needed and point to a new therapeutic venue for potentially treating and/or modulating autoimmune disease.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Primary biliary cirrhosis (PBC) is a chronic, progressive autoimmune liver disease characterized by immune-mediated destruction of biliary epithelial cells (BEC) that line the intrahepatic small bile ducts. The destruction of these cells leads to hepatic fibrosis and eventual liver failure [1,2]. Because the aetiology of PBC remains unclear, the goal in treating early-stage PBC is to slow the progression of disease and alleviate symptoms. Accumulated evidence by our group has demonstrated that autoreactive T cells that proliferate in response to mitochondrial autoantigens play a critical role in the destruction of the BECs [3–7]. Additionally, CD8+ T cells have been shown to predominate in the cellular infiltrates of portal tracts [3]. This conclusion has been supported by our recent work using a transgenic mouse model in which mice express a dominant negative form of transforming growth factor-β receptor type II specifically in T cells (dnTGF-βRII).

dnTGF-βRII mice developed spontaneously periductular aggregates of lymphocytes in liver in association with serum reactivity to the E2 component of pyruvate dehydrogenase complex (PDC-E2), similar to human PBC [8]. Further, the adoptive transfer of CD8+ T cells from dnTGF-βRII mice to Rag1−/− recipients results in PBC-like liver damage, while CD4+ T cells are less able to cause portal inflammation [9]. These findings suggest that CD8+ T cells are important in mediating the destruction of the small bile duct cells in PBC. Therefore, we hypothesized that the selective regulation of the hepatic CD8+ T cells should reduce liver damage in dnTGF-βRII mice.

β-Glucosylceramide (GC) is a naturally occurring glycosphingolipid. GC has been shown to function as a ‘fine-tuning factor’ in several mouse models of immune-mediated disorders [10,11]. The administration of GC protects against chronic graft-versus-host disease in mice due to an increase in the intrahepatic CD4/CD8 T cell ratio and decreased CD8+ T cell trapping [12]. In addition, in a colitis animal model, GC treatment alters the expression of the ganglioside GM1, a key marker of cell membrane lipid rafts on CD4+ and CD8+ T cells, and alleviates immune-mediated colitis [13]. In this work, we studied the effect of GC on autoimmune cholangitis in dnTGF-βRII mice. Our results demonstrate that treatment with GC from 6 weeks to 24 weeks of age reduces lymphocyte infiltration in liver and alleviates cholangitis in dnTGF-βRII mice; autoreactivate CD8+ T cells in the liver were decreased significantly in GC-treated mice when compared to control-treated mice.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Animals

dnTGF-βRII mice were bred on a C57BL/6 background at the University of California Davis animal facility. All mice were fed a sterile rodent helicobacter medicated dosing system (three-drug combination) diet (Bio-Serv, Frenchtown, NJ, USA) and maintained in individually ventilated cages under specific pathogen-free conditions. Experiments were performed following approval from the University of California Animal Care and Use Committee.

GC treatment

β-Glucosylceramide (GluCer; GC), Fig. 1, extracted from soy bean (Avanti Polar Lipids, Alabaster, AL, USA), was dissolved in ethanol and then emulsified in phosphate-buffered saline (PBS). Sixteen 6-week-old dnTGF-βRII mice were assigned randomly to two groups (GC treatment and PBS controls). Mice were treated with a daily intraperitoneal (i.p.) injection of GC (1·5 µg GC in 100 µl PBS), while control mice received a daily i.p. injection of 100 µl PBS. During all experiments mice were fed their normal diet, as above, and maintained in individually ventilated cages under specific pathogen-free conditions. Animals were killed 18 weeks after initiation of treatment, i.e. at 24 weeks of age. Liver tissue was collected for pathological evaluation and flow cytometry.

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Figure 1. Structure of soybean β-glucosylceramide.

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Anti-mitochondrial antibodies (AMA)

Serum AMA were quantified via an enzyme-linked immunosorbent assay (ELISA) method using recombinant PDC-E2 [14,15]. One microgram recombinant PDC-E2 antigen in 100 µl carbonate buffer (pH 9·6) was coated onto 96-well ELISA plates at 4°C overnight. Plates were washed with PBS containing 0·05% Tween-20 (PBST) (FisherBiotech, Fair Lawn, NJ, USA), then blocked with 200 µl of 1% bovine serum albumin (BSA) in PBS for 1 h at room temperature. One hundred µl of diluted sera (1:200) was added to each well and incubated at room temperature for 1 h. Plates were washed with PBST at least three times. One hundred µl of horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin G + A + M (Zymed, San Diego, CA, USA) diluted (1:3000) in PBS with 1% BSA was added to each well and incubated for 1 h at room temperature. Plates were rewashed and 100 µl of tetramethylbenzidine (TMB) peroxidase substrate (BD Biosciences) was added to each well. Optical density (OD) was read at 450 nm. Previously defined positive and negative controls were included with each test.

Histopathological evaluation of liver

Immediately after killing, the liver was harvested, fixed in 10% buffered formalin at room temperature for 2 days, embedded in paraffin and cut into 4-µm sections. The liver sections were deparaffinized, stained with haematoxylin and eosin (H&E) and evaluated using light microscopy.

Flow cytometry

Mononuclear cells were isolated from liver tissue using density gradient centrifugation with Accu-Paque (Accurate Chemical & Scientific Corp., Westbury, NY, USA). Anti-mouse CD16/32 (clone 93, Biolegend, San Diego, CA, USA) was used to block the Fc receptor. The mononuclear cells were stained with fluorochrome-conjugated antibodies including Alexa Fluor 750-conjugated anti-T cell receptor (TCR)-β (clone 57-597; eBiosciences, San Diego, CA, USA), Alexa Fluor 647-conjugated anti-CD19 (clone eBio1 3; eBiosciences), peridinin chlorophyll (PerCP)-conjugated anti-CD4 (clone RM4-5; Biolegend), fluorescein isothiocyanate (FITC)-conjugated anti-CD8a (clone 53-6·7; Biolegend) and phycoerythrin (PE)-conjugated anti-natural killer (NK)1·1 (clone PK136; BD-PharMingen, San Diego, CA, USA). Stained cells were analysed using a fluorescence activated cell sorter (FACScan) flow cytometer (BD Bioscience) that was upgraded by Cytec Development (Fremont, CA, USA), which allows for five-colour analysis. Data were analysed with cellquest software (BD Bioscience). Known positive and negative controls were used throughout.

Statistical analysis

The data are presented as the mean ± standard error of the mean. Two-sample comparisons were analysed using the two-tailed unpaired t-test. A value of P < 0·05 was considered statistically significant.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Treatment with GC alleviates liver inflammation in dnTGF-βRII transgenic mice

As expected, we detected high-titre AMAs readily in the sera of dnTGF-βRII transgenic mice [8]. Interestingly, 18 weeks after initiation of the GC treatment (or at 24 weeks of age) there was a slight decrease of sera AMAs in the GC-treated mice compared to controls (Fig. 2); however, this was not statistically significant. None the less, there was a dramatic improvement in the portal inflammation in the GC-treated group compared to the controls (Fig. 3a). For example, there were only mild or minimal liver cellular infiltrates in GC-treated mice compared to moderate liver inflammation in 40% of the control-treated animals (Fig. 3b). The lymphocytic infiltrates in liver of dnTGF-βRII mice contain both CD4+ and CD8+ T cells but are dominated by CD8+ T cells (Fig. 4a). We therefore analysed the composition of the intrahepatic lymphoid cell population within the liver of transgenic mice following 18 weeks of GC treatment. As shown in Fig. 4, the percentage of CD8+ T cells was decreased significantly in mice treated with GC compared to control dnTGF-βRII mice. The frequency of CD8+ cells in the NK1·1- TCR-β+ T cell gate was 46·73 ± 3·13% in the GC-treated mice and 58·44 ± 4·34% (P < 0·05) in the control mice (Fig. 4a). Importantly, the absolute number of intrahepatic CD8+ T cells was also decreased significantly in GC-treated mice compared to controls (1·73 ± 0·13 × 106versus 2·91 ± 0·48 × 106; P < 0·05; Fig. 4b). In contrast, no significant change in the number of CD4+ T cells (1·06 ± 0·21 × 106versus 0·99 ±  0·10 × 106) was observed (Fig. 4b). Additionally, GC-treated dnTGF-βRII mice exhibited a significant increase in the hepatic CD4/CD8 T cell ratio compared to controls (0·64 ± 0·10 versus 0·35 ± 0·05, respectively, P < 0·05; Fig. 4b). Furthermore, the number of CD19+ B cells and CD4+ NK T cells (TCR-β+NK1·1+) remained the same following GC treatment (Fig. 5). These results suggest that GC targets specifically intrahepatic CD8+ T cells.

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Figure 2. Sera anti-mitochondrial antibodies by enzyme-linked immunosorbent assay. There are no significant differences in optical density between β-glucosylceramide-treated and control mice. Threshold values are defined by mean ± 3 standard deviations for control animals.

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image

Figure 3. β-Glucosylceramide (GC) treatment alleviates liver inflammation. (a) Liver pathology. Light microscopy of haematoxylin and eosin-stained section of GC-treated and control mouse livers demonstrates deduced infiltration of lymphoid cells in portal tracts. (b) Liver inflammation and portal inflammation was scored after 18 weeks of GC (n = 9) or phosphate-buffered saline (n = 7) treatment: 0 for none, 1 for minimal, 2 for mild and 3 for moderate inflammation. *P < 0·05.

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image

Figure 4. Effects of β-glucosylceramide (GC) treatment on the hepatic CD8+ T cell population. (a) Flow cytometric analysis of the CD4+ and CD8+ T cell populations isolated from liver tissue, displayed as dot plots and gated on the lymphocyte population (upper panels) or the T cell receptor (TCR)-β+ natural killer (NK)1·1- T cell population (lower panels). The numbers in the dot plots are the percentage of cells in the specific quadrants (lower panels). (b) The number of lymphoid cell, and the CD4/CD8 ratio of cells, isolated from the liver of dominant negative form of transforming growth factor-β receptor type II (dnTGF-βRII) mice treated with or without GC (GC, n = 9; phosphate-buffered saline, n = 7). *P < 0·05; Tg+, dnTGF-βRII transgenic mice; horizontal bars represent the mean cell number.

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image

Figure 5. Profile of lymphoid cell populations in the liver of β-glucosylceramide (GC)-treated mice. Hepatic mononuclear cells were isolated from dominant negative form of transforming growth factor-β receptor type II (dnTGF-βRII) mice treated with or without GC. The cells were stained with T cell receptor (TCR)-β-, CD4-, CD8-, CD19- and natural killer (NK)1·1-specific antibodies and then analysed by flow cytometry. The data reflecting CD19+ B cells and CD4+NK1·1+ TCR-β+ cells are presented (GC, n = 9; phosphate-buffered saline, n = 7).

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Intrahepatic CD8+ T cell activation is attenuated by GC administration

To address further how GC administration inhibits the liver infiltration of CD8+ T cells in dnTGF-βRII mice, we analysed CD44 expression on the infiltrating lymphocytes. As seen in Fig. 6, the number of memory CD44high CD8+ T cells decreased significantly following GC treatment compared to the control mice (GC, 0·91 ± 0·09 × 106; PBS, 1·51 ±  0·22 × 106; P < 0·05). However, the number of memory CD44high CD4+ T cells was not different between the two groups (Fig. 6).

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Figure 6. Number of T cells with an activated memory phenotype (CD44high). Hepatic mononuclear cells were isolated from dominant negative form of transforming growth factor-β receptor type II (dnTGF-βRII) mice treated with or without β-glucosylceramide (GC); cells were stained with T cell receptor (TCR)-β-, CD4-, CD8-, natural killer (NK)1·1- and CD44-specific antibodies and analysed by flow cytometry. The data reflecting the number of CD4+ CD44high T cells and CD8+ CD44high T cells (GC, n = 9; phosphate-buffered saline, n = 7), *P < 0·05; horizontal bars represent mean cell number.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Sphingolipids are key components of cellular membranes and distributed widely in plants and animal [16]. There are structural variations among the sphingolipids of different organisms based on polarity and backbones; sphingolipids contribute structurally to membrane lipid bilayer and membrane lipid rafts formation. In addition, they also have diverse cellular functions, such as mediating cell-to-cell recognition and communication and modulating cell signal transmission [17,18]. Thus, they are essential for the development and growth of organisms and have been implicated in a number of diseases [19,20].

In mammalian cells, sphingolipids are classified into two major groups, sphingomyelin and glycosphingolipids [21]. Most glycosphingolipids are synthesized from glucosylation of ceramide to form glucosylceramide which, in turn, serves as the source of complex glycosylated sphingolipids (e.g. gangliosides GM3 and GM1) [21,22]. Structurally, glucosylceramide has a sphingosine and ceramide backbone with one or more sugar residues at the 1-hydroxyl position in a β-glycosidic linkage. Glucosylceramide used in this study is a natural glycosphingolipid extraction from soybean, which is comprised primarily of ceramide with 4, 8-sphingadiene (d18:2Δ4,Δ8) and α-hydroxypalmitic acid (Fig. 1). Soybean β-glucosylceramides (GluCer, GC) have a different backbone from those of mammals; mammals have a delta 4-double bond (occasionally a delta 8-double bond), and also may manifest alpha-hydroxy fatty acids [23].

Administration of GC has been shown to reduce the inflammatory response in several mouse models of autoimmune disease. Additionally, both the reduced trapping of CD8+ lymphocytes in liver and the alleviation of concanavalin A (ConA)-induced liver damage have been observed in GC-treated mice [24]. GC administration improves the glucose tolerance and hepatic steatosis seen in diabetic Cohen rats [11]. GC treatment has also been shown to alter the distribution of CD8+ T cells and NK T cells in the liver and augment anti-hepatitis B virus (HBV) immune responses [25]. However, there are no data that address the role of GC in the regulation of inflammation in an autoimmune liver disease model. Therefore, in this study, we investigated the effect of GC on an autoimmune cholangitis using dnTGF-βRII mice. Our data demonstrate that administration of GC ameliorates inflammation significantly in murine autoimmune cholangitis, as measured by an increased intrahepatic CD4/CD8 ratio and a decreased number of intrahepatic activated CD44high CD8+ T cells. We did not observe any changes in other lymphocyte populations such as CD4+ T cells, NK1·1+ TCR-β+ NK T cells and CD19+ B cells. Furthermore, the autoantibody titre in the serum (against anti-mitochondrial antigens) was not changed significantly in GC-treated mice compared to the control mice. These immunological alterations suggest that GC targets hepatic CD8+ T cells selectively, but not other lymphocytes in our murine cholangitis model.

A possible mechanism underlying the beneficial effects by GC treatment has been suggested by the interaction between GC and lipid rafts on immune cells. Lipid rafts, also called membrane microdomains, are specialized membrane domains that are highly dynamic submicroscopic assemblies. Furthermore, lipid rafts are enriched in sphingolipids and cholesterol and provide a particularly ordered lipid environment. The alteration of lipid rafts after T cell activation has been demonstrated extensively. Following activation by cross-linking of various receptors on T cells, the microdomains of lipid rafts become larger and more stable than those in resting T cells, often being attached to the cytoskeleton [26,27]. Lipid rafts play a critical role in the maturation, activation and immunological synapse formation in CD4+ and CD8+ T cells [28–32]. In addition, GC may potentially be a ligand for NK T cells [33,34]. Following binding to its receptor, GC inhibits the α-GalCer-mediated activation of NK T cells in vitro[35] and affects the function of NK T cells via the alteration of lipid rafts [34].

Our present data in a murine autoimmune cholangitis model demonstrate that the frequency, absolute number and the activation of intrahepatic CD8+ T cells were affected by GC treatment. Interestingly, GC had no significant effect on NK1·1+ TCR-β+ NK T cells in liver, in either frequency or cell number. Compatible with our previous findings, GC treatment in trinitrobenzene sulphonic acid-induced murine colitis led to a decrease in the expression of GM1, a ganglioside, on CD8+ T cells but not on NK T cells [13]. GM1 is one of the most common markers used for the identification of lipid rafts. Furthermore, GM1 accumulates in the immunological synapse, and its expression is up-regulated on activated CD8+ T cells in the lungs of respiratory syncytial virus-infected mice [36]. Therefore, the mechanism underlying the GC-mediated amelioration of cholangitis in dnTGF-βRII mice may occur through a reaction with the lipid rafts on CD8+ T lymphocytes that inhibit CD8+ T cell activation.

There are striking similarities in this model of autoimmune cholangitis, the dnTGF-βRII mouse, compared to humans with PBC. These similarities include the presence of AMA, elevated levels of inflammatory cytokines as well as the presence of CD4 and CD8 T cells in liver infiltrates. In both patients and mice, CD8 T cells predominate in portal tracts [8,9,37]. There remain significant gaps in the treatment of human PBC. For example, the use of immunosuppressive agents in humans with PBC has been disappointing, and there are virtually no data on the use of biological therapy. We are particularly intrigued by the data obtained herein with GC and suggest that further research and, in particular, focus on the relationship of GC with the lipid rafts may lead to a better understanding of mechanisms and potential usage of this dietary therapy. Clearly, future studies will need an emphasis on dose–response relationships and also a cleaner dissection of mechanisms of action.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

We thank Katsunori Yoshida for AMA detection and Thomas P. Kenny for technical support. We are grateful to Nikki Phipps for manuscript preparation. This work was supported financially by National Institute of Health grants DK39588 and DK074768 (M. E. G.); DK077961 (Z. X. L.) and UCD Center for Health and Nutrition Research Pilot Grant (Z. X. L.).

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References