Yuki Moritoki

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.)

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.)

Hepatic IL-17 responses in human and murine primary biliary cirrhosis.

The emergence of new regulatory and pro-inflammatory immune cell subsets and cytokines dictates the need to re-examine the role of these subsets in various diseases involving the immune system. IL-17 has been recently identified as a key cytokine involved in numerous autoimmune processes. However, its role in liver autoimmune diseases remains unclear. Primary biliary cirrhosis (PBC) is characterized histologically by autoreactive CD4 and CD8 T cells surrounding damaged bile ducts. CD4(+) T cells are a major source of IL-17, which compose a distinct T helper subset (Th17). Thus we set out to determine the role of IL-17 in both human and a murine model of PBC in a liver-targeted manner. Our data demonstrate an increase in the frequency of IL-17(+) lymphocytic infiltration in liver tissues from PBC patients and those with other liver dysfunctions as compared to healthy livers. IL-2 receptor alpha knockout mice, a recently identified murine model of human PBC, also demonstrate marked aggregations of IL-17-positive cells within portal tracts and increased frequencies of Th17 cells in the liver compared to the periphery. Interestingly, CD4(+) T cells from livers of normal C57BL/6J mice also secreted higher levels of IL-17 relative to those from spleens, indicating a preferential induction of Th17 cells in liver tissues. Importantly, C57BL/6J cocultures of splenic CD4(+) T cells and liver non-parenchymal cells increased IL-17 production approximately 10-fold compared to T cells alone, suggesting a role of the liver microenvironment in Th17 induction in cases of liver autoimmunity and other liver inflammatory diseases.

Loss of tolerance in C57BL/6 mice to the autoantigen E2 subunit of pyruvate dehydrogenase by a xenobiotic with ensuing biliary ductular disease.

There have been important advances in defining effector mechanisms for several human autoimmune diseases. However, for most human autoimmune diseases, the induction stage is less well defined and there are very few clues on etiology. Our laboratory has focused on defining the molecular basis of autoantibody recognition and epitope modification in primary biliary cirrhosis (PBC). Our work has demonstrated that antibodies to mitochondria, the hallmark of disease, are directed against a very conserved site of pyruvate dehydrogenase, the E2 subunit of pyruvate dehydrogenase (PDC‐E2). We have also demonstrated that several chemical xenobiotics, chosen based on quantitative structural activity relationship analysis and rigorous epitope analysis, when coupled to the lysine residue that normally binds the lipoic acid cofactor of PDC‐E2, reacts as well or better to PBC sera than native autoantigen. In the present studies, we immunized C57BL/6 mice with one such xenobiotic, 2‐octynoic acid, coupled to bovine serum albumin and we followed the mice for 24 weeks. Animals were studied for appearance of histologic lesions as well as appearance of antibodies to PDC‐E2, serum levels of tumor necrosis factor–α and interferon‐γ, and splenic and liver lymphoid phenotyping by flow cytometry. Mice immunized with 2‐octynoic acid manifest autoimmune cholangitis, typical mitochondrial autoantibodies, increased liver lymphoid cell numbers, an increase in CD8+ liver infiltrating cells, particularly CD8+ T cells that coexpress CD44, and finally an elevation of serum tumor necrosis factor–α and interferon‐γ. Conclusion: these data provide a persuasive argument in favor of an environmental origin for human PBC. (HEPATOLOGY 2008.)

Deletion of interleukin‐12p40 suppresses autoimmune cholangitis in dominant negative transforming growth factor β receptor type II mice

Our laboratory has reported that mice that express a dominant negative form of transforming growth factor β receptor restricted to T cells (dnTGFβRII) develop an inflammatory biliary ductular disease with elevated serum levels of interleukin (IL)‐12p40 and other proinflammatory cytokines and antimitochondrial autoantibodies (AMAs) closely resembling human primary biliary cirrhosis (PBC). We have used this mouse model to address the potential mechanisms of immunomodulation of liver disease by creating two unique genetic strains: IL‐12p40 knockout (KO)‐dnTGFβRII mice and IFN‐γ KO‐dnTGFβRII mice. The two colonies of genetically modified mice—and, for purposes of controls, the dnTGFβRII mice—were monitored for liver immunopathology, AMAs, and intrahepatic cytokine production. Disease expression in the IFN‐γ KO‐dnTGFβRII mice, including liver immunopathology, were similar to those of dnTGFβRII mice, whereas the IL‐12p40 KO‐dnTGFβRII mice had a dramatic reduction in histological autoimmune cholangitis and significant decreases in levels of intrahepatic proinflammatory cytokines, but similar levels of AMAs compared with dnTGFβRII controls. Conclusion: These data indicate that in this mouse model of PBC, signaling by way of IL‐12p40 is an essential requirement for the development of autoimmune cholangitis. The results of these studies will play an important role in identifying pathways and reagents that will selectively inhibit IL‐12 signaling for the outlining of future therapeutic strategies for human PBC. (HEPATOLOGY 2009.)

Evaluation of differential gene expression by microarray analysis in small and large cholangiocytes isolated from normal mice

Aims: We have shown that large and small cholangiocytes, which reside primarily in large and small intrahepatic bile ducts, respectively, have different functions and responses to injuries. However, there are no systematic studies of the molecular differences between small and large cholangiocytes, which would explain cholangiocyte heterogeneity. To evaluate the differential gene expression between small and large cholangiocytes, microarray analysis was performed.

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.)

Biochemical and immunologic effects of rituximab in patients with primary biliary cirrhosis and an incomplete response to ursodeoxycholic acid.

The aim of this study was to determine the safety and potential efficacy of B‐cell depletion with the anti‐CD20 monoclonal antibody rituximab in patients with primary biliary cirrhosis (PBC) and an incomplete response to ursodeoxycholic acid (UDCA). This open‐label study enrolled six patients with PBC and incomplete responses to UDCA to be treated with 2 doses of 1000 mg rituximab separated by 2 weeks and followed for 52 weeks. The primary endpoints were safety and changes in B‐cell function. Two patients received only 1 dose of rituximab, one due to activation of latent varicella and the other due to a viral upper respiratory infection. Serum levels of total IgG, IgM, and IgA as well as anti‐mitochondrial autoantibodies (AMAs) IgA and IgM decreased significantly from baseline by 16 weeks and returned to baseline levels by 36 weeks. Stimulation of B cells with CpG produced significantly less IgM at 52 weeks after treatment compared with B cells at baseline. In addition, transient decreases in memory B‐cell and T‐cell frequencies and an increase in CD25high CD4+ T cells were observed after treatment. These changes were associated with significant increases in mRNA levels of FoxP3 and transforming growth factor‐β (TGF‐β) and a decrease in tumor necrosis factor‐α (TNF‐α) in CD4+ T cells. Notably, serum alkaline phosphatase levels were significantly reduced up to 36 weeks following rituximab treatment. Conclusion: These data suggest that depletion of B cells influences the induction, maintenance, and activation of both B and T cells and provides a potential mechanism for treatment of patients with PBC with an incomplete response to UDCA. (HEPATOLOGY 2012)

Myeloid STAT3 Inhibits T Cell-Mediated Hepatitis by Regulating T Helper 1 Cytokine and Interleukin-17 Production

BACKGROUND & AIMS T cell-mediated hepatitis is a leading cause of acute liver failure; there is no effective treatment, and the mechanisms underlying its pathogenesis are obscure. The aim of this study was to investigate the immune cell-signaling pathways involved-specifically the role of signal transducer and activator of transcription 3 (STAT3)-in T cell-mediated hepatitis in mice. METHODS T cell-mediated hepatitis was induced in mice by injection of concanavalin A (Con A). Mice with myeloid cell-specific and T-cell-specific deletion of STAT3 were generated. RESULTS STAT3 was activated in myeloid and T cells following Con A injection. Deletion of STAT3 specifically from myeloid cells exacerbated T-cell hepatitis and induced STAT1-dependent production of a T helper cell (Th)1 cytokine (interferon [IFN]-gamma) and to a lesser extent of Th17 cytokines (interleukin [IL]-17 and IL-22) in a STAT1-independent manner. In contrast, deletion of STAT3 in T cells reduced T cell-mediated hepatitis and IL-17 production. Furthermore, deletion of IFN-gamma completely abolished Con A-induced T-cell hepatitis, whereas deletion of IL-17 slightly but significantly reduced such injury. In vitro experiments indicated that IL-17 promoted liver inflammation but inhibited hepatocyte apoptosis. CONCLUSIONS Myeloid STAT3 activation inhibits T cell-mediated hepatitis via suppression of a Th1 cytokine (IFN-gamma) in a STAT1-dependent manner, whereas STAT3 activation in T cells promotes T-cell hepatitis to a lesser extent, via induction of IL-17. Therefore, activation of STAT3 in myeloid cells could be a novel therapeutic strategy for patients with T-cell hepatitis.