Reversible phospho-Smad3 signalling between tumour suppression and fibrocarcinogenesis in chronic hepatitis B infection.

Transforming growth factor (TGF)-ß, type I receptor (TßRI) and c-Jun N-terminal kinases (JNK) phosphorylate Smad3 differentially to create 2 isoforms phosphorylated (p) at the COOH-terminus (C) or at the linker region (L) and regulate hepatocytic fibrocarcinogenesis. This study aimed to compare the differences between how hepatitis B virus (HBV) infection affected hepatocytic Smad3 phosphorylated isoforms before and after anti-viral therapy. To clarify the relationship between Smad3 phosphorylation and liver disease progression, we studied 10 random patients in each stage of HBV-related fibrotic liver disease (F1-4) and also 10 patients with HBV-associated HCC. To examine changes in phosphorylated Smad3 signalling before and after anti-HBV therapies, we chose 27 patients with chronic hepatitis B who underwent baseline and follow-up biopsies at 52 weeks from the start of nucleoside analogue treatments (Lamivudine 100 mg daily or Telbivudine 600 mg daily). Fibrosis stage, inflammatory activity and phosphorylated Smad3 positivity in the paired biopsy samples were compared. Hepatocytic pSmad3C signalling shifted to fibrocarcinogenic pSmad3L signalling as the livers progressed from chronic hepatitis B infection to HCC. After nucleoside analogue treatment, serum alanine aminotransferase (ALT) and HBV-DNA levels in 27 patients with HBV-related chronic liver diseases were decreased dramatically. Decrease in HBV-DNA restored pSmad3C signalling in hepatocytes, while eliminating prior fibrocarcinogenic pSmad3L signalling. Oral nucleoside analogue therapies can suppress fibrosis and reduce HCC incidence by successfully reversing phosphorylated Smad3 signalling; even liver disease progressed to cirrhosis in chronic hepatitis B patients.

Retinoic acids up-regulate functional eosinophil-driving receptor CCR3.

Eotaxins and their receptor CCR3 have a definitive role for tissue accumulation of eosinophils both under homeostatic and pathologic conditions. However, physiological stimuli that can up-regulate CCR3 in blood-derived human eosinophils have not been recognized. As a prior gene microarray study revealed up-regulation of CCR3 in eosinophils stimulated with retinoic acids (RAs), the expression of functional CCR3 was examined. We found that 9-cis RA and all-trans RA (ATRA) significantly induced surface CCR3 expression regardless of the presence of IL-3 or IL-5. Pharmacological manipulations with receptor-specific agonists and antagonists indicated that retinoic acid receptor-α activation is critical for CCR3 up-regulation. RA-induced CCR3 was associated with its functional capacity, in terms of the calcium mobilization and chemotactic response to eotaxin-1 (CCL11). Our study suggests an important role of vitamin A derivatives in the tissue accumulation of eosinophils.

Beta-glucosylceramide ameliorates liver inflammation in murine autoimmune cholangitis.

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-beta receptor (dnTGF-betaRII) 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 beta-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-betaRII 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 CD44(high) 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.

Induction of autoimmune cholangitis in non-obese diabetic (NOD).1101 mice following a chemical xenobiotic immunization.

Our laboratory has suggested that loss of tolerance to pyruvate dehydrogenase (PDC-E2) leads to an anti-mitochondrial antibody response and autoimmune cholangitis, similar to human primary biliary cirrhosis (PBC). We have suggested that this loss of tolerance can be induced either via chemical xenobiotic immunization or exposure to select bacteria. Our work has also highlighted the importance of genetic susceptibility. Using the non-obese diabetic (NOD) congenic strain 1101 (hereafter referred to as NOD.1101 mice), which has chromosome 3 regions from B6 introgressed onto a NOD background, we exposed animals to 2-octynoic acid (2OA) coupled to bovine serum albumin (BSA). 2OA has been demonstrated previously by a quantitative structural activity relationship to react as well as or better than lipoic acid to anti-mitochondrial antibodies. We demonstrate herein that NOD.1101 mice immunized with 2OA-BSA, but not with BSA alone, develop high titre anti-mitochondrial antibodies and histological features, including portal infiltrates enriched in CD8(+) cells and liver granulomas, similar to human PBC. We believe this model will allow the rigorous dissection of early immunogenetic cause of biliary damage.

Generation of functionally distinct B lymphocytes from common myeloid progenitors.

Current models of adult haematopoiesis propose that haematopoietic stem cells (HSCs) differentiate into common lymphoid (CLP) and common myeloid (CMP) progenitors and establish an early separation between myeloid and lymphoid lineages. Nevertheless, the developmental potential of CMP-associated B cells suggests the existence of alternate pathways for B lymphopoesis. The aim of this study was to compare the developmental and functional properties of CMP- and CLP-derived B cells. While both populations matured through pro-B cell and transitional B cell intermediates in the bone marrow and spleen, respectively, following transfer into irradiated mice, mature CMP- and CLP-derived B cells exhibit distinct functional responses. Specifically, CMP-derived B cells did not respond to mitogenic stimulation to the same degree as their CLP-derived counterparts and secrete lower levels of IgM and the inflammatory cytokines such as interleukin (IL)-6 and IL-10. Together, these data suggest the existence of multiple pathways for generating functionally distinct B cells from bone marrow precursors.

The role of CD11c(+) hepatic dendritic cells in the induction of innate immune responses.

The role of the liver in the initiation and maintenance of tolerance is a critical immune function that involves multiple lineages of immune cells. Included within these populations are liver dendritic cells (DCs). Although there has been significant work on the phenotypic and functional roles of splenic and bone marrow dendritic cells, as well as their subsets, comparable studies in liver have often been difficult. To address this issue we have isolated, from C57BL/6 mice, relatively pure populations of DCs and compared phenotype and function to the data from spleen using flow cytometry, cell sorter assisted purification and culture, morphology by cytospin and May-Giemsa staining, cell cycle progression, antigen uptake, cytokine production and allo-activation potential. natural killer (NK)1.1(-)CD11c(+) liver DC subsets (conventional DCs, T cell receptor (TcR)beta(-)NK1.1(-)CD11c(+)B220(-) and plasmacytoid DCs, TcRbeta(-)NK1.1(-)CD11c(+)B220(+)) efficiently endocytose dextran and produce significant levels of tumour necrosis factor (TNF)-alpha, interleukin (IL)-6 and IL-12 p40 in response to Toll-like receptor (TLR) ligands, with responses higher than splenic DCs. There is also a differential capability of hepatic DCs to respond to innate signals. Indeed, CD11c(+) hepatic DCs have a greater capacity to respond to innate stimulation but are less capable of inducing CpG activated-allogeneic T cells. These data suggest that hepatic dendritic cells function as a critical bridge between innate and adaptive immunity and are capable of inducing stronger innate responses with a lower capacity for allo-stimulation than splenic dendritic cells. These properties of liver dendritic cells contribute to their unique role in the induction of tolerance.