Kazuhito Kawata

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;)

Overexpression of microRNA-21 is associated with elevated pro-inflammatory cytokines in dominant-negative TGF-β receptor type II mouse.

Dominant-negative TGF-β receptor II (dnTGF-βRII) mice spontaneously develop an autoimmune cholangitis resembling human primary biliary cirrhosis (PBC). Interestingly, the dominant-negative TGF-β receptor is expressed by both CD4(+) and CD8(+) T cells and leads to greatly reduced (but not absent) TGF-β signaling resulting in T cell intrinsic cell mediated autoimmunity. However, the mechanisms of the T cell dysregulation remain unclear. Recently it has been shown that TGF-β signaling is intimately involved with miRNA biogenesis and control. Herein we show that lack of T cell TGF-β signaling leads to down regulation of T cell miRNAs but up-regulation of the key inflammatory miRNA 21. Furthermore, the expression of miR-21 from hepatic effector CD8(+) T cells is significantly higher than in the same subsets isolated from spleen and mesenteric lymph nodes of the dnTGF-βRII mice. Previous studies indicate that miR-21 increases the synthesis of IFN-γ and IL-17A by T cells and suppresses apoptosis via programmed cell death protein 4 (PDCD4). Data presented herein demonstrate that transfecting w.t. B6 T cell subsets with miR-21 resulted in up-regulation of the inflammatory cytokines TNF-α and IFN-γ, thus partly replicating the dnTGF-βRII T cell phenotype. In conclusion, these data suggest miR-21 plays a critical role in the production of pro-inflammatory cytokines in dnTGF-βRII mice, which could be a contributing factor for the development of the organ-specific autoimmune cholangitis and colitis in this murine model of human PBC.

Identification of Potential Cytokine Pathways for Therapeutic Intervention in Murine Primary Biliary Cirrhosis

Primary biliary cirrhosis (PBC) is considered a model autoimmune disease, with the most highly directed and specific autoantibody in both murine and human autoimmunity, the anti-mitochondrial autoantibody (AMA). However, therapeutic advances in this disease have lagged behind. Herein we have taken advantage of our unique model of murine PBC in which mice immunized with 2-octynoic acid coupled to BSA (2OA-BSA), a compound identified by quantitative structure activity relationships (QSAR) of human AMA binding, develop an intense inflammatory cholangitis with striking similarities to humans with PBC. In particular, we have constructed several unique gene-deleted mice, including mice deleted of IL-12p40, IL-12p35, IFN-γ, IL-23p19, IL-17A, IL-17F and IL-22, immunized these animals with 2OA-BSA and followed the natural history of immunopathology to identify key pathways that might provide clues for successful therapy. Our data indicate that whereas both IL-12/Th1 and IL-23/Th17 are involved in cholangitis, it is the IL-12/Th1 signaling pathway that elicits pathology. In fact, deletion of IFN-γ prevents disease and suppresses autoantibodies. Importantly, deletion of the Th17 cytokines IL-17A and IL-22, but not IL-17F, reduces biliary damage; IL-17A-knockout mice have reduced levels of anti-mitochondrial antibody. We further demonstrate that the production of IFN-γ is significantly decreased in the liver of IL-23p19−/−, IL-17A−/− and IL-22−/− mice compared with controls. However, the ability of T cells to produce IFN-γ was not affected in Th17 cytokine-deficient mice. Our data indicate that a deficient Th17 pathway suppresses the accumulation of IFN-γ producing cells in liver during the early phase of cholangitis. In conclusion, whereas IFN-γ has a pivotal role in the early events involved in the pathogenesis of autoimmune cholangitis induced by 2OA-BSA, the IL-23/Th17 pathway potentiates the effects of IL-12/IFN-γ-mediated immunopathology.

Clonality, activated antigen‐specific CD8+ T cells, and development of autoimmune cholangitis in dnTGFβRII mice

There are several murine models described with features similar to human primary biliary cirrhosis (PBC). Among these models, the one which has the closest serologic features to PBC is a mouse with a T‐cell‐restricted expression of the dominant negative transforming growth factor β receptor type II (dnTGFβRII). Our work has demonstrated that CD8+ T cells from dnTGFβRII mice transfer autoimmune cholangitis to Rag1−/− recipients. However, it remained unclear whether the autoimmune cholangitis was secondary to an intrinsic function within CD8+ T cells or due to the abnormal TGFβR environment within which CD8+ T cells were generated. To address this mechanistic issue, we used our dnTGFβRII, OT‐I/Rag1−/−, OT‐II/Rag1−/− mice and in addition generated OT‐I/dnTGFβRII/Rag1−/−, and OT‐II/dnTGFβRII/Rag1−/− mice in which the entire T‐cell repertoire was replaced with ovalbumin (OVA)‐specific CD8+ or CD4+ T cells, respectively. Importantly, neither the parental OT‐I/dnTGFβRII/Rag1−/− mice and/or OT‐II/dnTGFβRII/Rag1−/− mice developed cholangitis. However, adoptive transfer demonstrated that only transfer of CD8+ T cells from dnTGFβRII mice but not CD8+ T cells from OT‐I/Rag1−/− mice or from OT‐I/dnTGFβRII/Rag1−/− mice transferred disease. These data were not secondary to an absence of CD4+ T cell help since a combination of CD8+ T cells from OT‐I/dnTGFβRII/Rag1−/− and CD4+ T cells from OT II/dnTGFβRII/Rag1−/− or CD8+ T cells from OT‐I/dnTGFβRII/Rag1−/− with CD4+ T cells from OT‐II/Rag1−/− mice failed to transfer disease. Conclusion: Defective TGFβRII signaling, in addition to clonal CD8+ T cells that target biliary cells, are required for induction of autoimmune cholangitis. (Hepatology 2013;53:1094–1104)

Enhanced Hepatic Nrf2 Activation After Ursodeoxycholic Acid Treatment in Patients with Primary Biliary Cirrhosis

The cytoprotective mechanisms of ursodeoxycholic acid (UDCA) in primary biliary cirrhosis (PBC) have not been fully clarified. UDCA has some antioxidant properties. Nuclear factor-E2-related factor-2 (Nrf2) plays a critical role in protecting a variety of tissues against oxidative stress. Therefore, to investigate the potential antioxidant effects of UDCA in PBC, we determined the intracellular status of both oxidant stress and antioxidant defenses in paired pre- and posttreatment liver biopsies from 13 PBC patients by immunodetection of 8-hydroxydeoxyguanosine (8-OHdG), Nrf2-, and Nrf2-mediated antioxidant proteins. After UDCA treatment, the number of 8-OHdG-positive hepatocytes or bile duct cells decreased with improvement of hepatic injury. The hepatic levels of both total and phosphorylated Nrf2 protein were increased, along with upregulation of nuclear phosphorylated Nrf2 expression in bile duct cells. In addition, the levels of both thioredoxin (TRX) and thioredoxin reductase 1 (TrxR1) protein were increased in the liver after UDCA. The upregulation of hepatic TRX or TrxR1 protein expression positively correlated with that of total Nrf2 protein expression. In conclusion, UDCA treatment can enhance hepatic Nrf2 activation and upregulate hepatic TRX and TrxR1 protein expression. Hepatic Nrf2 activation may play a role in the therapeutic response to UDCA in PBC.

The immunophysiology and apoptosis of biliary epithelial cells: primary biliary cirrhosis and primary sclerosing cholangitis.

Biliary epithelial cells (BECs) provide the first line of defense against lumenal microbes in the biliary system. BECs express a variety of pathogen recognition receptors and can activate several intracellular signaling cascades to initiate antimicrobial defenses, including production of several anti-microbial peptides, cytokines, chemokines, and adhesion molecules. BECs also secrete immunoglobulin A and interact with other cells through expression and release of adhesion molecules and immune mediators. Recently, several reports suggest a correlation between apoptosis and autoimmunity through ineffective clearance of self-antigens. Primary biliary cirrhosis (PBC) is a slowly progressive, autoimmune cholestatic liver disease characterized by highly specific antimitochondrial antibodies (AMAs) and the specific immune-mediated destruction of BECs. We have demonstrated that the AMA self-antigen, namely the E2 subunit of the pyruvate dehydrogenase complex, is detectable in its antigenically reactive form within apoptotic blebs from human intrahepatic biliary epithelial cells and activates innate immune responses. Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by inflammation and the presence of concentric fibrosis of intrahepatic and/or extrahepatic bile ducts, eventually leading to cirrhosis. However, apoptosis does not appear to play a central role in PSC. Despite both diseases involving immune-mediated injury to bile ducts, apoptosis occurs more commonly overall in PBC where it likely plays a unique role.

Mincle and human B cell function.

C-type lectin receptors are pattern recognition receptors that are critical for autoimmunity and the immune response. Mincle is a C-type lectin receptor expressed by a variety of antigen presenting cells including macrophages, neutrophils, dendritic cells and B cells; a variety of stimuli including stress are known to induce the expression of Mincle. Mincle is an FcRγ-associated activation receptor that senses damaged cells and upon ligation induces activated macrophages to produce inflammatory cytokines. Recently, while several studies have reported that Mincle plays an important role in macrophage responses to fungal infection its function on B cells remains to be defined. In efforts to elucidate the function of Mincle expressed by B cells, we studied the expression of Mincle on subsets of B cells and analyzed cytokines and synthesized immunoglobulin upon ligation of Mincle. The expression of Mincle on CD27-CD19(+) naïve B cells is significantly higher than CD27 + CD19(+) memory B cells. The stimulation of TLR9 ligand induced Mincle expression on B cells. Furthermore, co-stimulation of TLR9 and Mincle ligand reduced IgG and IgA production from B cells without a significant change in the inflammatory cytokines TNF-α, IL-6, IL-8 and IL-10. Our data identifies Mincle as a potentially critical player in human B cell responses.

Differential expression of hepatic apurinic/apyrimidinic endonuclease 1, a DNA repair enzyme, in chronic hepatitis

AIM To determine hepatic expression of apurinic/apyrimidinic endonuclease 1 (APE-1) and 8-hydroxydeoxyguanosine (8-OHdG) in patients with chronic hepatitis B and C. METHODS Liver biopsies were obtained from 27 patients with chronic hepatitis B virus (HBV), 30 with chronic hepatitis C virus (HCV), 6 with autoimmune hepatitis (AIH), and 6 with primary biliary cirrhosis (PBC). Normal liver tissue was obtained from surgical resection specimens of four patients. Hepatic APE-1 protein and mRNA expression were assayed by Western blot and by real-time polymerase chain reaction, respectively. Hepatocellular APE-1 and 8-OHdG expression were determined by immunohistochemistry. RESULTS The staining intensity of hepatocellular nuclear APE-1 was lower in the HBV group than in the other groups (P < 0.05). Hepatic APE-1 protein levels were reduced in the HBV group relative to the other groups. Hepatic APE-1 mRNA levels were also lower in the HBV group. The proportion of hepatocytes with 8-OHdG-positive nuclei was increased in the HCV, AIH and PBC groups (P < 0.05), but not in the HBV group. Hepatocellular nuclear APE-1 levels were positively correlated with hepatocellular 8-OHdG levels in both the HBV and HCV groups (HBV, r = 0.34, P < 0.05; HCV, r = 0.54, P < 0.01). CONCLUSION An imbalance between oxidative DNA damage and APE-1 expression may contribute to hepatocarcinogenesis in chronic viral hepatitis.

Improved Serum Alpha-Fetoprotein Levels after Iron Reduction Therapy in HCV Patients

Background and Aims. To examine the changes in serum alpha-fetoprotein (AFP) levels after iron reduction by therapeutic phlebotomy in chronic hepatitis C patients. Methods. This retrospective study included 26 chronic hepatitis C patients. The patients were developed iron depletion by repeated therapeutic phlebotomies. Results. Iron reduction therapy significantly reduced the median level of serum AFP from 13 to 7 ng/mL, ALT from 96 to 50 IU/L, gamma-glutamyl transpeptidase (GGT) from 55 to 28 IU/L, and ferritin from 191 to 10 ng/mL (P < 0.001 for each). The rate of decline in the AFP level correlated positively only with that in GGT (r = 0.695, P = 0.001), although a spurious correlation was observed between the rates of decline for AFP and ALT. The AFP level normalized (<10 ng/mL) posttreatment in eight (50%) of 16 patients who had elevated pretreatment AFP levels. Normalized post-treatment ALT and GGT levels were seen in 12% (3 of 26) and 39% (7 of 18) of the patients, respectively. Multivariate analysis identified a post-treatment GGT level of <30 IU/L as an independent factor associated with post-treatment AFP normalization (odds ratio, 21; 95% confidence interval, 1.5–293; P = 0.024). Conclusions. Iron reduction by therapeutic phlebotomy can reduce serum AFP and GGT levels in chronic hepatitis C patients.

Critical role of CREBH‐mediated induction of transforming growth factor β2 by hepatitis C virus infection in fibrogenic responses in hepatic stellate cells

Mechanisms of hepatic fibrogenesis induced by hepatitis C virus (HCV), one of the leading causes of liver fibrosis, are not fully understood. We studied transcriptional up‐regulation of transforming growth factor β (TGF‐β), especially TGF‐β2, which is mediated by activation of liver‐enriched transcription factor cAMP‐responsive element‐binding protein, hepatocyte specific (CREBH) triggered by HCV infection and its functional significance for induction of profibrogenic phenotypes by interaction of HCV‐infected cells with hepatic stellate cells (HSCs). Compared to TGF‐β1, expression of TGF‐β2 mRNA was induced faster and to a higher level upon HCV infection. Serum TGF‐β2 levels in hepatitis C patients were higher compared to those in healthy individuals and were positively correlated with hepatic fibrosis stages F0‐F2. TGF‐β2 promoter activity was decreased and increased, respectively, by silencing and overexpression of CREBH. CREBH recognition sites were identified in the TGF‐β2 promoter. CREBH binding to the promoter and its increase in cells expressing HCV Core‐NS2 were shown by gel mobility shift and chromatin immunoprecipitation, respectively. The active form of CREBH was detectable in HCV‐infected chimeric mice with human livers and cells expressing HCV proteins. Involvement of CREBH in HCV‐induced fibrogenic response was further demonstrated in the CREBH null‐mutant mouse model. Fibrogenic phenotypes were assessed using co‐cultures of HCV‐infected cells and HSCs. Expressions of fibrogenic factors and TGF‐β1 increasing in the co‐cultures was prevented by TGF‐β2‐ or CREBH silencing. Conclusion: CREBH was identified as a key positive regulator of TGF‐β2 transcription in HCV‐infected cells. TGF‐β2 released from infected cells potentially contributes to cross‐induction of TGF‐β in an autocrine manner through its own signaling pathway, leading to an increase in fibrogenic responses in adjacent HSCs. (Hepatology 2017;66:1430–1443).