Ya-Hui Chuang

Liver-targeted and peripheral blood alterations of regulatory T cells in primary biliary cirrhosis

CD4+CD25high regulatory T cells (Tregs) play a critical role in self‐tolerance, as seen in murine autoimmunity. Studies on Tregs in human autoimmunity have focused primarily on peripheral blood samples. A study targeting diseased tissue should identify direct relationships between Tregs and autoimmunity. Peripheral blood samples were collected from 91 patients with primary biliary cirrhosis (PBC), 28 immediate relatives, and 41 healthy controls, and Treg frequencies were determined as a percentage of CD4+CD25high T cells in CD4+TCR‐αβ+ T cells. A tissue‐targeted determination of frequency and distribution of FoxP3+ Tregs was also performed on 90 different liver tissue specimens exhibiting PBC (n = 52), chronic hepatitis C (CHC) (n = 30), and autoimmune hepatitis (AIH) (n = 8). Treg suppression studies were performed on 50 PBC patients and 27 controls. Patients with PBC demonstrated a relative reduction of Tregs compared with controls (P < .0002). Interestingly, a deficiency in CD4+CD25+ Tregs was also found in the daughters and sisters of PBC patients compared with controls (P < .0007). However, functional studies did not reveal a global PBC Treg defect. The level of FoxP3‐expressing Tregs was markedly lower in affected PBC portal tracts compared with CHC and AIH (P < .001). In addition, the CD8+T cell/FoxP3+ Treg ratio was significantly higher in livers of late‐stage PBC compared with those of CHC (P < .001) and early‐stage AIH (P < .001). In conclusion, these data provide support for a genetic modulation of Treg frequency and illustrate the role Tregs play in the loss of tolerance in PBC. (HEPATOLOGY 2006;43:729–737.)

Anti-Mitochondrial Antibodies and Primary Biliary Cirrhosis in TGF-β Receptor II Dominant-Negative Mice

Primary biliary cirrhosis (PBC) is an autoimmune disease of the liver, characterized by lymphocytic infiltrates in portal tracts, selective destruction of biliary epithelial cells, and anti-mitochondrial Abs (AMAs). The elucidation of early events in the induction of tissue inflammation and autoimmunity in PBC has been hampered by the cryptic onset of the disease, the practical limitations in accessing the target tissue, and the lack of a suitable animal model. We demonstrate in this study that a mouse transgenic for directed expression of a dominant-negative form of TGF-β receptor type II (dnTGFβRII), under the direction of the CD4 promoter, mimics several key phenotypic features of human PBC, including spontaneous production of AMAs directed to the same mitochondrial autoantigens, namely PDC-E2, BCOADC-E2, and OGDC-E2. The murine AMAs also inhibit PDC-E2 activity. Moreover, there is lymphocytic liver infiltration with periportal inflammation analogous to the histological profile in human PBC. Additionally, the serum cytokine profile of affected mice mimics data in human PBC. The concomitant presence of these immunopathological features in the transgenic mice suggests that the TGF-βRII pathway is implicated in the pathogenesis of PBC. Finally, these data point away from initiation of autoimmunity by mechanisms such as molecular mimicry and more toward activation of an intrinsically self-reactive T cell repertoire in which necessary regulatory T cell influences are lacking.

IL‐2 receptor α−/− mice and the development of primary biliary cirrhosis

Recently, we identified a child born with a genetic deficiency of IL‐2 receptor α (IL‐2Rα, CD25) expression who had several clinical manifestations of primary biliary cirrhosis (PBC). In addition, there has been suggestive evidence in both patients with PBC and their first‐degree relatives that a deficiency of regulatory T cells (Tregs) is an integral component for susceptibility to PBC. Based on these observations, we generated IL‐2Rα/CD25 deficient (IL‐2Rα−/−) mice and wild‐type littermate controls and followed them longitudinally for the natural history of liver immunopathology and appearance of antimitochondrial antibodies (AMAs). The analyses included immunohistochemical staining of liver and portal tract infiltrates as well as FACS profiles of lymphoid subpopulations in liver and spleen. In addition, serum cytokine profiles were quantitated. Importantly, IL‐2Rα−/−, but not littermate controls, develop portal inflammation and biliary ductular damage similar to human patients with PBC. CD4+ and CD8+ T cells predominate among portal cell infiltrates and sera reflect a Th1 cytokine bias with increased levels of IFN‐γ, TNF‐α, IL‐2 and IL‐12p40. Of importance is the finding that the IL‐2Rα−/− mice not only develop significantly increased serum levels of IgG and IgA, but they also develop AMAs with specificity for PDC‐E2, which maps to the inner lipoyl domain of the autoantigen, all characteristics which are hallmarks of human PBC. In conclusion, the IL‐2Rα−/− mice should facilitate studies of the early events in PBC and especially the tantalizing connection between Treg deficiency and autoimmunity specifically directed to mitochondrially located PDC‐E2 and subsequent biliary ductular cell damage. (HEPATOLOGY 2006;44:1240–1249.)

The immunobiology of Henoch–Schönlein purpura☆

Henoch-Schönlein purpura (HSP) is a common but enigmatic systemic small vessel vasculitis that primarily affects children. Although the etiology of this disease is unknown, there are tantalizing clues on the natural history and immunopathogenesis. This article reviews these clues including aspects of disease-associated pathogens, immune regulation, with a focus on IgA, and finally the immunogenetic background of host. We also present a hypothetical model for the development of HSP and submit that this paradigm will be a generic one for similar vasculopathies.

Natural killer T cells exacerbate liver injury in a transforming growth factor β receptor II dominant‐negative mouse model of primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is an organ‐specific autoimmune liver disease characterized by the presence of antimitochondrial antibodies and the destruction of small intrahepatic bile ducts with portal inflammation. In previous studies, we reported that both CD1d expression and the frequency of CD1d‐restricted natural killer T (NKT) cells were increased in the livers of patients with PBC. To define a specific role of CD1d‐restricted NKT cells in the pathogenesis of PBC, particularly early events, we investigated the function of hepatic CD1d‐restricted NKT cells in our transforming growth factor β (TGF‐β) receptor II dominant‐negative (dnTGFβRII) mouse model of PBC. We generated CD1d−/− and CD1d+/− dnTGFβRII mice and performed a comparative study of liver immunopathology. We report herein that these dnTGFβRII mice demonstrate a massive increase of hyperactive CD1d‐restricted NKT cells within the hepatic tissues. CD1d−/−dnTGFβRII mice, which lack CD1d‐restricted CD1d‐restricted NKT cells, exhibit significantly decreased hepatic lymphoid cell infiltrates and milder cholangitis compared with CD1d+/−dnTGFβRII mice. Interestingly, there was a significant increase in the production of interferon‐γ in hepatic CD1d‐restricted NKT cells activated by α‐galactosylceramide in young but not older dnTGFβRII mice, suggesting an age‐dependent role of CD1d‐restricted NKT cells. Conclusion: These data demonstrate that CD1d‐restricted NKT cells in dnTGFβRII mice are a critical factor in liver injury. (HEPATOLOGY 2008.)

Adoptive transfer of CD8+ T cells from transforming growth factor beta receptor type II (dominant negative form) induces autoimmune cholangitis in mice

We recently reported that mice with a T cell–restricted expression of a dominant negative form of transforming growth factor β receptor type II (dnTGFβRII) spontaneously develop autoimmune cholangitis that resembles human primary biliary cirrhosis (PBC), including antimitochondrial antibodies (AMAs) and extensive portal CD4+ and CD8+ lymphocytic infiltrates. On the basis of these data, we performed a series of experiments to determine whether the pathology was secondary to direct dnTGFβRII disruption of the liver and/or alternatively the appearance of autoreactive T cells. First, using dnTGFβRIIRag1−/− mice, we noted a normal hepatic and biliary structure. Hence, we performed a rigorous series of adoptive transfer studies, transferring Ly5.1+ unfractionated spleen cell CD4+ or CD8+ T cells from dnTGFβRII mice into B6/Rag−/− (Ly 5.2) recipients. In unmanipulated dnTGFβRII mice, there was a marked increase in CD4+ and CD8+ T cell biliary infiltrates with AMA. Indeed, B6/Rag−/− recipients of dnTGFβRII unfractionated cells develop features of liver disease similar to PBC, suggesting that splenic loss of self‐tolerance alone is sufficient to cause disease in this model and therefore that there is no specific abnormality in the biliary targets required for appearance of disease. More importantly, adoptive transfer of CD8+ but not CD4+ T cells into B6/Rag−/− mice led to liver histopathology remarkably similar to PBC, emphasizing a prominent role for CD8 T cell–mediated pathogenesis. In contrast, B6/Rag−/− recipients of CD4+ T cells from dnTGFβRII mice predominantly developed inflammatory bowel disease associated with higher levels of serum interferon γ and tumor necrosis factor α. Conclusion: These data suggest that in this model of PBC, autoreactive CD8+ cells destroy bile ducts. (HEPATOLOGY 2008.)

Innate immunity and primary biliary cirrhosis: Activated invariant natural killer T cells exacerbate murine autoimmune cholangitis and fibrosis

Murine models of autoimmunity allow the study of the earliest events in disease pathogenesis. Our laboratory has developed a xenobiotic induced model of primary biliary cirrhosis (PBC) following immunization of mice with 2‐octynoic acid coupled to bovine serum albumin (2‐OA‐BSA), an antigen selected following quantitative structure‐activity relationship analysis of the E2 subunit of the pyruvate dehydrogenase complex (PDC‐E2), the immunodominant autoantigen of PBC. Recent data in humans with PBC has suggested that a major component of liver pathology is due to activation of innate immunity. We took advantage of our 2‐OA‐BSA model and immunized mice with and without the addition of α‐galactosylceramide (α‐GalCer), an invariant natural killer T cell activator. Importantly, we report herein that 2‐OA‐BSA‐immunized mice exposed to α‐GalCer develop a profound exacerbation of their autoimmune cholangitis, including significant increases in CD8+ T‐cell infiltrates, portal inflammation, granuloma formation, and bile duct damage. Furthermore, such mice produce increased levels of antimitochondrial antibodies and have evidence of fibrosis, a feature not previously reported in the murine models of PBC. Conclusion: Our data suggests a primary role of innate immunity in the exacerbation of autoimmune cholangitis and also become a logical explanation for the recurrence of PBC following liver transplantation in the absence of major histocompatability complex compatibility. We submit that PBC begins with loss of tolerance to PDC‐E2 and a multilineage antimitochondrial response in which autoreactive CD8+ T cells are critical. However, the perpetuation of disease and its exacerbation will also be modulated by innate immune mechanisms. (HEPATOLOGY 2011;)

AMA production in primary biliary cirrhosis is promoted by the TLR9 ligand CpG and suppressed by potassium channel blockers

We previously reported that peripheral blood mononuclear cells (PBMCs) from patients with primary biliary cirrhosis (PBC) produce significantly higher levels of polyclonal IgM than controls after exposure to CpG. Furthermore, the prevalence and unusually high levels of antimitochondrial antibodies (AMAs) in patients with PBC suggest a profound loss of B cell tolerance. We have addressed the issue of whether CpG will promote the production of AMAs and whether new experimental agents that inhibit the lymphocyte potassium channels Kv1.3 and KCa3.1 can suppress CpG‐mediated B cell activation and AMA production. PBMCs were stimulated with and without CpG and were subsequently analyzed for phenotype, including expression of TLR9, CD86, and KCa3.1 concurrent with measurements of AMA and responses to a control antigen, tetanus toxoid, in supernatants. Additionally, K+ channel expression on B cells from PBC patients and controls was studied using whole‐cell patch‐clamp technology. In patients with PBC, CpG induces secretion of AMAs in PBMCs and also up‐regulates B cell expression of TLR9, CD86, and KCa3.1. Additionally, K+ channel blockers suppress secretion of AMA without a reduction of CpG‐B–enhanced IgM production. Furthermore, there is diminished up‐regulation of TLR9 and CD86 without affecting proliferation of B cells, B cell apoptosis, or viability. Conclusion: These data suggest that the hyperresponsiveness of B cells in PBC accelerates B cell–mediated autoimmunity. (HEPATOLOGY 2007;45:314–322.)

The level of IgA antibodies to human umbilical vein endothelial cells can be enhanced by TNF-α treatment in children with Henoch–Schönlein purpura

Anti‐endothelial cell antibodies (AECA) have been found to play an important role in many vascular disorders. In order to determine the presence of AECA in children with Henoch–Schönlein purpura (HSP), and to elucidate the pathogenic and clinical value of their measurement in this disease, AECA were detected by immunofluorescence staining and a human umbilical vein endothelial cell (HUVEC)‐based enzyme‐linked immunosorbent assay (ELISA) in 20 children with HSP, 10 children with juvenile rheumatoid arthritis (JRA) without vasculitis and 10 normal healthy children. Antibodies against another endothelial cells, human dermal microvascular endothelial cells (HMVEC‐d) were also detected by cell‐based ELISA. In some experiments, we compared the binding activity of antibodies to HUVEC with and without tumour necrosis factor‐α (TNF‐α) or interleukin‐1 (IL‐1) pretreatment. Patients with acute onset of HSP had higher serum levels of IgA antibodies, both against HUVEC and against HMVEC‐d, than healthy controls (P = 0·001, P = 0·008, respectively). Forty‐five per cent of patients had positive IgA AECA to HUVEC, and 35% had positive IgA AECA to HMVEC‐d. The titres of IgA antibodies to HUVEC paralleled the disease activity. After TNF‐α treatment, the values of IgA AECA to HUVEC in HSP patients were significantly increased (P = 0·02). For IgG and IgM AECA, there was no difference between HSP patients and controls (P = 0·51, P = 0·91). Ten JRA children without vasculitis had no detectable IgG, IgM or IgA AECA activity. The results of this study showed that children with HSP had IgA AECA, which were enhanced by TNF‐α treatment. Although the role of these antibodies is not clear, IgA AECA provide another immunological clue for the understanding of HSP.

CD4+CD25+ Foxp3+ Regulatory T Cells Protect against T Cell-Mediated Fulminant Hepatitis in a TGF-β-Dependent Manner in Mice

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.