Weici Zhang

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.)

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.

Deficiency in regulatory T cells results in development of antimitochondrial antibodies and autoimmune cholangitis

There have been several descriptions of mouse models that manifest select immunological and clinical features of autoimmune cholangitis with similarities to primary biliary cirrhosis in humans. Some of these models require immunization with complete Freunds adjuvant, whereas others suggest that a decreased frequency of T regulatory cells (Tregs) facilitates spontaneous disease. We hypothesized that antimitochondrial antibodies (AMAs) and development of autoimmune cholangitis would be found in mice genetically deficient in components essential for the development and homeostasis of forkhead box 3 (Foxp3)+ Tregs. Therefore, we examined Scurfy (Sf) mice, animals that have a mutation in the gene encoding the Foxp3 transcription factor that results in a complete abolition of Foxp3+ Tregs. At 3 to 4 weeks of age, 100% of animals exhibit high‐titer serum AMA of all isotypes. Furthermore, mice have moderate to severe lymphocytic infiltrates surrounding portal areas with evidence of biliary duct damage, and dramatic elevation of cytokines in serum and messenger RNAs encoding cytokines in liver tissue, including tumor necrosis factor α, interferon‐γ, interleukin (IL)‐6, IL‐12, and IL‐23. Conclusion: The lack of functional Foxp3 is a major predisposing feature for loss of tolerance that leads to autoimmune cholangitis. These findings reflect on the importance of regulatory T cells in other murine models as well as in patients with primary biliary cirrhosis. (HEPATOLOGY 2008.)

Therapeutic effect of cytotoxic T lymphocyte antigen 4/immunoglobulin on a murine model of primary biliary cirrhosis

Collectively, the data in both humans and murine models of human primary biliary cirrhosis (PBC) suggest that activated T cells, particularly CD8 T cells, play a critical role in biliary cell destruction. Under physiological conditions, T‐cell activation involves two critical signals that involve the major histocompatibility complex and a set of costimulatory molecules, which include a receptor on T cells termed cytotoxic T lymphocyte antigen 4 (CTLA‐4). Germane to the studies reported herein, signaling by CTLA‐4 has the potential to modulate costimulation and induce inhibitory signals. In this study, we have taken advantage of our well‐defined murine model of PBC, in which mice are immunized with 2‐octynoic acid coupled to bovine serum albumin (2OA‐BSA), leading to the production of high‐titer antimitochondrial autoantibodies (AMAs) and portal cellular infiltrates. To investigate the potential of CTLA‐4‐Ig (immunoglobulin) as an immunotherapeutic agent, we treated mice both before and after induction of autoimmune cholangitis. First, we demonstrate that CTLA‐4‐Ig treatment, begun 1 day before 2OA‐BSA immunization, completely inhibits the manifestations of cholangitis, including AMA production, intrahepatic T‐cell infiltrates, and bile duct damage. However, and more critically, treatment with CTLA‐4‐Ig, initiated after the development of autoimmune cholangitis in previously immunized mice, also resulted in significant therapeutic benefit, including reduced intrahepatic T‐cell infiltrates and biliary cell damage, although AMA levels were not altered. Conclusion: These data suggest that an optimized regimen with CTLA‐4‐Ig has the potential to serve as an investigative therapeutic tool in patients with PBC. (HEPATOLOGY 2013)

B-cell depletion with anti-CD20 ameliorates autoimmune cholangitis but exacerbates colitis in transforming growth factor-β receptor II dominant negative mice

The treatment of primary biliary cirrhosis (PBC) with conventional immunosuppressive drugs has been relatively disappointing and there have been few efforts in defining a role for the newer biological agents useful in rheumatoid arthritis and other systemic autoimmune diseases. In this study we took advantage of transforming growth factor‐β (TGF‐β) receptor II dominant negative (dnTGF‐βRII) mice, a mouse model of autoimmune cholangitis, to address the therapeutic efficacy of B‐cell depletion using anti‐CD20. Mice were treated at either 4‐6 weeks of age or beginning at 20‐22 weeks of age with intraperitoneal injections of anti‐CD20 every 2 weeks. We quantitated B‐cell levels in all mice as well as antimitochondrial antibodies (AMA), serum and hepatic levels of proinflammatory cytokines, and histopathology of liver and colon. In mice whose treatment was initiated at 4‐6 weeks of age, anti‐CD20 therapy demonstrated a significantly lower incidence of liver inflammation associated with reduced numbers of activated hepatic CD8+ T cells. However, colon inflammation was exacerbated. In contrast, in mice treated at 20‐22 weeks of age, anti‐CD20 therapy had relatively little effect on either liver or colon disease. As expected, all treated animals had reduced levels of B cells, absence of AMA, and increased levels in sera of tumor necrosis factor alpha (TNF‐α), interleukin 6 (IL‐6), and chemokine (C‐C motif) ligand (CCL2) (monocyte chemoattractant protein 1 [MCP‐1]). Conclusion: These data suggest potential usage of anti‐CD20 in early PBC resistant to other modalities, but raise a cautionary note regarding the use of anti‐CD20 in inflammatory bowel disease. (HEPATOLOGY 2009.)

B Cells Suppress the Inflammatory Response in a Mouse Model of Primary Biliary Cirrhosis

BACKGROUND & AIMS Mice that express a dominant-negative form of transforming growth factor-beta receptor restricted to T cells (dnTGF-betaRII) develop antimitochondrial antibodies and liver inflammation similar to human primary biliary cirrhosis. METHODS To address the role of B cells in this model of primary biliary cirrhosis, we bred B cell-deficient mice (Igmu(-/-)) with dnTGF-betaRII mice, creating Igmu(-/-)dnTGF-betaRII mice, and compared the resulting disease phenotype with that of dnTGF-betaRII mice (controls). We also performed adoptive transfer of dnTGF-betaRII CD8(+) splenocytes, with or without B cells, to 8-week-old female Rag-1(-/-) mice to assess the role of B cells in the inflammatory response. RESULTS The B cell-deficient Igmu(-/-)dnTGF-betaRII mice unexpectedly developed a more severe form of cholangitis than controls (dnTGF-betaRII mice) and had a significantly greater frequency of activated CD4(+) and CD8(+) T cells in the liver. They also had reduced frequency of Foxp3(+) regulatory T cells in the hepatic CD4(+) T-cell population and natural killer (NK) T cells (NK1.1(+) CD3(+)) in hepatic inflammatory cell infiltrates. The Igmu(-/-)dnTGF-betaRII mice had increased levels of proinflammatory cytokines (tumor necrosis factor-alpha and interleukin-6) and developed a more severe form of colitis than controls. Adoptive transfer of CD8(+) splenocytes from dnTGF-betaRII mice and peritoneal cavity-derived, but not spleen-derived, CD19(+) B cells into Rag-1(-/-) mice resulted in decreased amounts of liver inflammation and bile duct damage, compared with Rag-1(-/-) mice in which only CD8(+) splenocytes were transferred. CONCLUSION B cells have a suppressive effect on the inflammatory response in the dnTGF-betaRII model of primary biliary cirrhosis.

Deletion of interleukin (IL)‐12p35 induces liver fibrosis in dominant‐negative TGFβ receptor type II mice

Mice with a dominant‐negative transforming growth factor β receptor restricted to T cells (dnTGFβRII mice) develop an inflammatory biliary ductular disease that strongly resembles human primary biliary cirrhosis (PBC). Furthermore, deletion of the gene encoding interleukin (IL)‐12p40 resulted in a strain (IL‐12p40−/−dnTGFβRII) with dramatically reduced autoimmune cholangitis. To further investigate the role of the IL‐12 cytokine family in dnTGFβRII autoimmune biliary disease, we deleted the gene encoding the IL‐12p35 subunit from dnTGFβRII mice, resulting in an IL‐12p35−/− dnTGFβRII strain which is deficient in two members of the IL‐12 family, IL‐12 and IL‐35. In contrast to IL‐12p40−/− mice, the IL‐12p35−/−mice developed liver inflammation and bile duct damage with similar severity but delayed onset as the parental dnTGFβRII mice. The p35−/− mice also demonstrated a distinct cytokine profile characterized by a shift from a T‐helper 1 (Th1) to a Th17 response. Strikingly, liver fibrosis was frequently observed in IL‐12p35−/− mice. In conclusion, IL‐12p35−/− dnTGFβRII mice, histologically and immunologically, reflect key features of PBC, providing a useful generic model to understand the immunopathology of human PBC. (HEPATOLOGY 2013;)

Differential mechanisms in the pathogenesis of autoimmune cholangitis versus inflammatory bowel disease in interleukin-2Rα-/- mice

Interleukin‐2 (IL‐2) receptor α knockout (IL‐2Rα−/−) mice have a deficiency of CD25 and a corresponding functional defect in T regulatory cells (Tregs). These mice spontaneously develop portal inflammation with biliary ductular damage and colitis with features similar to human inflammatory bowel disease with T cell infiltrates in both the liver and colon. In humans, inflammatory bowel disease may be accompanied by primary sclerosing cholangitis (PSC), but seldom primary biliary cirrhosis (PBC). We hypothesized that the effector mechanism responsible for T cell infiltrates would differ for colon versus liver. To address this thesis, we developed three colonies of double‐knockout mice including IL‐2Rα−/− CD4−/−, IL‐2Rα−/− CD8−/−, and IL‐2Rα−/− T cell receptor (TCR)‐β−/−. Tissue immunopathology, body weight, and serum levels of cytokines, immunoglobulins, and anti‐mitochondrial antibodies (AMA) were assayed at 3 months of age. Relative to IL‐2Rα−/− mice, IL‐2Rα−/− CD4−/− mice had increased biliary ductular damage but reduced inflammation in the colon. In contrast, IL‐2Rα−/− CD8−/− mice had increased colon inflammation but markedly attenuated biliary ductular damage. Both IL‐2Rα−/− CD4−/− and IL‐2Rα−/− CD8−/− mice demonstrated elevated serum levels of tumor necrosis factor alpha (TNF‐α), interferon gamma (IFN‐γ), interleukin‐12p40 (IL‐12p40), and interleukin‐2 (IL‐2) compared with C57BL/6J controls, but only IL‐2Rα−/− CD8−/− mice had increased serum levels of immunoglobulin A (IgA), AMA and interleukin‐17 (IL‐17). Finally, and of importance, IL‐2Rα−/− TCR‐β−/− mice had abrogation of liver and colon pathological conditions and lacked AMA. In conclusion, on loss of Treg function in mice, CD8 T cells mediate biliary ductular damage whereas CD4 T cells mediate induction of colon‐specific autoimmunity. (HEPATOLOGY 2009;49:133‐140.)

Distinct from its canonical effects, deletion of IL-12p40 induces cholangitis and fibrosis in interleukin-2Rα−/− mice

The IL-12 family modulates T cell mediated autoimmune diseases and GWAS in PBC have suggested a critical role of IL-12 and its subunits in modulating portal inflammation. We have taken advantage of an aggressive model of portal inflammation and colitis in IL-2Rα(-/-) mice to study the specific role of IL-12 and, in particular, the immunobiology of p40(-/-)IL-2Rα(-/-) mice. Colonies of IL-2Rα(+/-), IL-2Rα(-/-) and p40(-/-)IL-2Rα(-/-) mice were studied for the natural history of immunopathology in liver and colon using histology and immunohistochemistry. Further, to focus on mechanisms, liver, spleen and mesenteric lymph node flow cytometry was employed to identify specific phenotypes; cytokine analysis on inflammatory cell populations was compared between groups. Finally, Real-Time PCR was used to focus on the genes involved in hepatic fibrosis. Surprisingly, p40(-/-)IL-2Rα(-/-) mice manifest more severe portal inflammation and bile duct damage, including signs of portal hypertension and liver fibrosis, but a significant reduction in colitis. Indeed, p40(-/-)IL-2Rα(-/-) mice reveal a profound hepatic CD8(+) T cell infiltrate, whose major component are effector memory cells as well as enhanced hepatic Th1 but reduced Th17 responses. These observations were confirmed by Real-Time PCR analysis of fibrosis-related genes in the liver. Distinct from its canonical effects, IL-12p40 plays a critical role in autoimmune cholangitis, including hepatic fibrosis. These data take on striking significance for any proposed human trials that modulate the IL-12p40 pathway in human PBC.