Murine Model Of Primary Biliary Cirrhosis Research Articles

Successful treatment of murine autoimmune cholangitis by parabiosis: Implications for hematopoietic therapy

There is a significant unmet need in the treatment of primary biliary cirrhosis (PBC) despite significant data on the effector pathways that lead to biliary duct damage. We focused attention on a murine model of PBC, the dominant negative transforming growth factor β receptor II (Tg) mice. To further define the pathways that lead to biliary pathology in these mice, we developed Tg mice deleted of CD4 cells (CD4−/−Tg).Interestingly, these mice developed more severe cholangitis than control Tg mice. These mice, which lack CD4 cells, manifested increased levels of IFN-γ produced by effector CD8 cells. It appears that increased cholangitis is due to the absence of CD4 Treg cells. Based on these data, we parabiosed CD4−/−Tg mice with established disease at 8–9 weeks of age with C57BL/6 control mice. Such parabiotic “twins” had a significant reduction in autoimmune cholangitis, even though they had established pathology at the time of surgery. We prepared mixed bone marrow chimera mice constructed from CD4−/−Tg and CD8−/− mice and not only was cholangitis improved, but a decrease in terminally differentiated CD8+ T effector cells in the presence of wild type CD4 cells was noted. In conclusion, “correcting” the CD4 T cell subset, even in the presence of pathogenic CD8 T cells, is effective in treating autoimmune cholangitis.

Aberrant TGF-β1 signaling contributes to the development of primary biliary cirrhosis in murine model

To investigate whether transforming growth factor-β1 (TGF-β1) signaling pathway is involved in the pathogenesis of primary biliary cirrhosis (PBC). A murine model of PBC was developed by injection of polyinosinic polycytidylic acids (poly I: C) in C57BL/6 mice, and the liver expressions of TGF β1, TGF-β receptor I (TβRI), TGF-β receptor II (TβRII), p-Smad2/3, monoclonal α-smooth muscle actin antibody (α-SMA) and α1 (I) collagen in the mouse model and control mice were evaluated by immunohistochemistry, immunoblotting and real-time polymerase chain reaction (RT-PCR). Lymphocyte subsets in liver were analyzed using flow cytometry. The mouse model had several key phenotypic features of human PBC, including elevated levels of alkaline phosphatase, antimitochondrial antibodies, portal bile ducts inflammation, and progressive collagen deposition. Compared with control mice, protein and mRNA levels of TGF β1, TβRI, TβRII, p-Smad2/3, α-SMA and α1 (I) collagen in liver (1.7 ± 0.4 vs 8.9 ± 1.8, 0.8 ± 0.2 vs 5.1 ± 1.5, 0.6 ± 0.01 vs 5.1 ± 0.1, 0.6 ± 0.3 vs 2.0 ± 0.3, 0.9 ± 0.4 vs 3.4 ± 0.6, 0.8 ± 0.4 vs 1.7 ± 0.3, 1.1 ± 1.2 vs 11.8 ± 0.6, P < 0.05), and the total number and percentage of CD4⁺ CD25⁺ FOXP3⁺ and CD8⁺ lymphocytes (0.01 ± 0.001 vs 0.004 ± 0.00, 0.12 ± 0.04 vs 0.52 ± 0.23, P < 0.01) were higher in the mouse model. TGFβ1 might play a dual role in the development of PBC: it suppresses inflammatory response but operates to enhance fibrogenesis. The aberrant activity of TGF-β1 signaling contributes to the development of PBC.

Plasma cells and the chronic nonsuppurative destructive cholangitis of primary biliary cirrhosis

There has been increased interest in the role of B cells in the pathogenesis of primary biliary cirrhosis (PBC). Although the vast majority of patients with this disease have anti-mitochondrial antibodies, there is no correlation of anti-mitochondrial antibody titer and/or presence with disease severity. Furthermore, in murine models of PBC, it has been suggested that depletion of B cells may exacerbate biliary pathology. To address this issue, we focused on a detailed phenotypic characterization of mononuclear cell infiltrates surrounding the intrahepatic bile ducts of patients with PBC, primary sclerosing cholangitis, autoimmune hepatitis, chronic hepatitis C, and graft-versus-host disease, including CD3, CD4, CD8, CD20, CD38, and immunoglobulin classes, as well as double immunohistochemical staining for CD38 and IgM. Interestingly, CD20 B lymphocytes, which are a precursor of plasma cells, were found in scattered locations or occasionally forming follicle-like aggregations but were not noted at the proximal location of chronic nonsuppurative destructive cholangitis. In contrast, there was a unique and distinct coronal arrangement of CD38 cells around the intrahepatic ducts in PBC but not controls; the majority of such cells were considered plasma cells based on their expression of intracellular immunoglobulins, including IgM and IgG, but not IgA. Patients with PBC who manifest this unique coronal arrangement were those with significantly higher titers of anti-mitochondrial antibodies. These data collectively suggest a role for plasma cells in the specific destruction of intrahepatic bile ducts in PBC and confirm the increasing interest in plasma cells and autoimmunity.

Innate Immunity and Primary Biliary Cirrhosis: Activated Invariant Natural Killer T Cells Exacerbate Murine Autoimmune Cholangitis and Fibrosis §Δ

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. 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.

Phenotypical and functional alterations of CD8 regulatory T cells in primary biliary cirrhosis

The mechanisms that lead to loss of tolerance in autoimmune disease have remained both elusive and diverse, including both genetic predisposition and generic dysregulation of critical mononuclear cell subsets. In primary biliary cirrhosis (PBC), patients exhibit a multilineage response to the E2 component of pyruvate dehydrogenase involving antibody as well as autoreactive CD4 and CD8 responses. Recent data from murine models of PBC have suggested that a critical mechanism of biliary destruction is mediated by liver-infiltrating CD8 cells. Further, the number of autoreactive liver-infiltrating CD4 and CD8 cells is significantly higher in liver than blood in patients with PBC. Based on this data, we have studied the frequencies and phenotypic characterization of both CD4 and CD8 regulatory T cell components in both patients with PBC and age–sex matched controls. Our data is striking and indicate that CD8 Treg populations from PBC patients, but not controls, have significant phenotypic alterations, including increased expression of CD127 and reduced CD39. Furthermore, in vitro induction of CD8 Tregs by incubation with IL10 is significantly reduced in PBC patients. Importantly, the frequencies of circulating CD4+CD25+ and CD8+ and CD28− T cell subpopulations are not significantly different between patients and controls. In conclusion, these data identify the CD8 Treg subset as a regulatory T cell subpopulation altered in patients with PBC.

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 α 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.