Pathogen infections and primary biliary cholangitis.

Primary biliary cholangitis (PBC) is a multi-factorial disease caused by the interaction of both genetic predisposition and environmental triggers. Bacterial infection has been investigated most intensively, both epidemiologically and experimentally, as a prime environmental aetiology in PBC. The association of recurrent history of urinary tract infection (UTI) with PBC has been frequently confirmed by several large-scale, case-control studies, despite variation in geographic area or case-finding methods. Escherichia coli is a predominant pathogen in most cases with UTI. Animal studies and molecular mimicry analysis between the human and E. coli E2 subunit of the 2-oxo-acid dehydrogenase complexes demonstrated that E. coli infection is a key factor in breaking immunological tolerance against the mitochondria, resulting in the production of anti-mitochondrial autoantibodies (AMA), the disease-specific autoantibodies of PBC. Novosphingobium aromaticivorans, a ubiquitous xenobiotic-metabolizing bacterium, is another candidate which may be involved in the aetiology of PBC. Meanwhile, improved environmental hygiene and increased prevalence of PBC, especially in males, may argue against the aetiological role of bacterial infection in PBC. Multiple mechanisms can result in the loss of tolerance to mitochondrial autoantigens in PBC; nonetheless, bacterial infection is probably one of the dominant pathways, especially in female patients. Notably, there is a rising prevalence of male patients with PBC. With increasing exposure to environmental xenobiotics in both genders, studies directed towards identifying the environmental culprit with systematically designed case-control studies are much needed to further determine the environmental factors and role of bacterial infections in PBC.

Endogenous interleukin-22 protects against inflammatory bowel disease but not autoimmune cholangitis in dominant negative form of transforming growth factor beta receptor type II mice.

During chronic inflammation, interleukin (IL)-22 expression is up-regulated in both CD4 and CD8 T cells, exerting a protective role in infections. However, in autoimmunity, IL-22 appears to have either a protective or a pathogenic role in a variety of murine models of autoimmunity and, by extrapolation, in humans. It is not clear whether IL-22 itself mediates inflammation or is a by-product of inflammation. We have taken advantage of the dominant negative form of transforming growth factor beta receptor type II (dnTGF-ßRII) mice that develop both inflammatory bowel disease and autoimmune cholangitis and studied the role and the biological function of IL-22 by generating IL-22(-/-) dnTGF-ßRII mice. Our data suggest that the influence of IL-22 on autoimmunity is determined in part by the local microenvironment. In particular, IL-22 deficiency exacerbates tissue injury in inflammatory bowel disease, but has no influence on either the hepatocytes or cholangiocytes in the same model. These data take on particular significance in the previously defined effects of IL-17A, IL-12p40 and IL-23p19 deficiency and emphasize that, in colitis, there is a dominant role of IL-23/T helper type 17 (Th17) signalling. Furthermore, the levels of IL-22 are IL-23-dependent. The use of cytokine therapy in patients with autoimmune disease has significant potential, but must take into account the overlapping and often promiscuous effects that can theoretically exacerbate inflammation.

T cell epitope immunotherapy ameliorates allergic responses in a murine model of shrimp allergy.

BACKGROUND: Shellfish allergy is one of the most common food hypersensitivities worldwide but allergen-specific immunotherapy for shellfish allergy is not yet available. We believe that T cell peptide-based immunotherapy holds the potential for modulating allergic responses without IgE cross-linking. OBJECTIVE: We sought to identify the immunodominant T cell epitopes of tropomyosin, the major shrimp allergen of Metapenaeus ensis (Met e 1), and to evaluate their therapeutic effects in a Balb/c mouse model of Met e 1 hypersensitivity. METHODS: T cell epitopes of Met e 1 were first identified based on the proliferation and cytokine responses of splenocytes isolated from Met e 1-sensitized Balb/c mice upon stimulation by 18 synthetic peptides that span the full-length Met e 1. The immunodominant T cell peptides identified were then fed orally to Met e 1-sensitized Balb/c mice twice a week for four weeks. Allergic responses, serological antibody levels, intestinal histology and systemic and local cytokine profiles were compared between the treated and the untreated groups. RESULTS: Six major Met e 1 T cell epitopes were identified. Mice treated with the T cell epitope peptide mixture demonstrated an amelioration of systemic allergic symptoms and a significant reduction in Th2-associated antibody and cytokine responses. These benefits were accompanied by a shift to a balanced Th1/Th2 response, induction of IgG2a antibodies possessing in vitro and in vivo blocking activities and the induction of regulatory T cell responses. CONCLUSIONS AND CLINICAL RELEVANCE: T cell epitope-based oral immunotherapy is effective in reducing allergic responses towards shrimp tropomyosin. This is a novel strategy for clinical management of shellfish allergy and is a model for mechanistic studies of oral immunotherapy.

Immunological potential of cytotoxic T lymphocyte antigen 4 immunoglobulin in murine autoimmune cholangitis.

Cytotoxic T lymphocyte antigen 4 (CTLA-4) immunoglobulin (Ig) is an important regulator of T cell activation and a fusion protein directed at CD80 and CD86; it blocks co-stimulatory signalling and T cell activation. We have taken advantage of a murine model of human primary biliary cirrhosis (PBC), mice expressing a transforming growth factor (TGF)-ß receptor II dominant negative (dnTGF-ßRII) transgene to address the potential therapeutic efficacy of CTLA-4 Ig. To mimic patients with PBC at different stages or duration of disease, we treated mice with either CTLA-4 Ig or control IgG three times weekly from 3 to 12 or 24 weeks of age, or from 12 to 24 weeks of age. CTLA-4 Ig treatment from 3 weeks of age significantly reduced liver inflammation to 12 weeks of age. Treatment initiated at 12 weeks of age also ameliorated the autoimmune cholangitis at 24 weeks of age. However, in mice treated at 3 weeks of age, suppression of liver inflammation was not sustained and colitis was aggravated when treatment was extended to 24 weeks of age. Our data indicate that, in dnTGF-ßRII mice, CTLA-4 Ig treatment has short-term beneficial effects on autoimmune cholangitis, but the effect varies according to duration of treatment and the time in which therapy was initiated. Further dissection of the events that lead to the reduction in therapeutic effectiveness of CTLA-4 Ig will be critical to determining whether such efforts can be applied to human PBC.

Innate immunity drives xenobiotic-induced murine autoimmune cholangitis.

Although primary biliary cirrhosis (PBC) is considered a model autoimmune disease, it has not responded therapeutically to traditional immunosuppressive agents. In addition, PBC may recur following liver transplantation, despite the absence of major histocompatibility complex (MHC) matching, in sharp contrast to the well-known paradigm of MHC restriction. We have suggested previously that invariant natural killer T (iNK T) cells are critical to the initiation of PBC. In this study we have taken advantage of our ability to induce autoimmune cholangitis with 2-octynoic acid, a common component of cosmetics, conjugated to bovine serum albumin (2-OA-BSA), and studied the natural history of pathology in mice genetically deleted for CD4 or CD8 following immunization with 2-OA-BSA in the presence or absence of α-galactosylceramide (α-GalCer). In particular, we address whether autoimmune cholangitis can be induced in the absence of traditional CD4 and CD8 responses. We report herein that CD4 and CD8 knock-out mice immunized with 2-OA-BSA/PBS or 2-OA-BSA/α-GalCer develop anti-mitochondrial antibodies (AMAs), portal infiltrates and fibrosis. Indeed, our data suggest that the innate immunity is critical for immunopathology and that the pathology is exacerbated in the presence of α-GalCer. In conclusion, these data provide not only an explanation for the recurrence of PBC following liver transplantation in the absence of MHC compatibility, but also suggest that effective therapies for PBC must include blocking of both innate and adaptive pathways.

Escherichia coli infection induces autoimmune cholangitis and anti-mitochondrial antibodies in non-obese diabetic (NOD).B6 (Idd10/Idd18) mice.

Several epidemiological studies have demonstrated that patients with primary biliary cirrhosis (PBC) have a higher incidence of urinary tract infections (UTI) and there is significant homology of the immunodominant mitochondrial autoantigen, the E2 component of the pyruvate dehydrogenase complex (PDC-E2), between mammals and bacteria. Previous work has demonstrated that non-obese diabetic (NOD).B6 Idd10/Idd18 infected with Novosphingobium aromaticivorans developed liver lesions similar to human PBC. It was postulated that the biliary disease was dependent upon the presence of the unique N. aro glycosphingolipids in activating natural killer T (NK T) cells. To address this issue, we infected NOD.B6 Idd10/Idd18 mice with either Escherichia coli, N. aro or use of a phosphate-buffered saline (PBS) vehicle control and serially followed animals for the appearance of liver pathology and anti-mitochondrial autoantibodies (AMA). Of striking importance, the biliary disease of E. coli-infected mice was more severe than N. Aro-infected mice and the titre of AMA was higher in E. coli-infected mice. Furthermore, the immunopathology did not correlate with the ability of bacterial extracts to produce antigen-dependent activation of NK T cells. Our data suggest that the unique glycosphingolipids of N. aro are not required for the development of autoimmune cholangitis. Importantly, the data highlight the clinical significance of E. coli infection in a genetically susceptible host, and we suggest that the appearance of autoimmune cholangitis is dependent upon molecular mimicry. These data highlight that breach of tolerance to PDC-E2 is probably the first event in the natural history of PBC in genetically susceptible hosts.

Human intrahepatic biliary epithelial cells engulf blebs from their apoptotic peers.

The phagocytic clearance of apoptotic cells is critical for tissue homeostasis; a number of non-professional phagocytic cells, including epithelial cells, can both take up and process apoptotic bodies, including the release of anti-inflammatory mediators. These observations are particularly important in the case of human intrahepatic biliary cells (HiBEC), because such cells are themselves a target of destruction in primary biliary cirrhosis, the human autoimmune disease. To address the apoptotic ability of HiBECs, we have focused on their ability to phagocytize apoptotic blebs from autologous HiBECs. In this study we report that HiBEC cells demonstrate phagocytic function from autologous HiBEC peers accompanied by up-regulation of the chemokines CCL2 [monocyte chemotactic protein-1 (MCP-1)] and CXCL8 [interleukin (IL)-8]. In particular, HiBEC cells express the phagocytosis-related receptor phosphatidylserine receptors (PSR), implying that HiBECs function through the 'eat-me' signal phosphatidylserine expressed by apoptotic cells. Indeed, although HiBEC cells acquire antigen-presenting cell (APC) function, they do not change the expression of classic APC function surface markers after engulfment of blebs, both with and without the presence of Toll-like receptor (TLR) stimulation. These results are important not only for understanding of the normal physiological function of HiBECs, but also explain the inflammatory potential and reduced clearance of HiBEC cells following the inflammatory cascade in primary biliary cirrhosis.

Epitope-specific anti-nuclear antibodies are expressed in a mouse model of primary biliary cirrhosis and are cytokine-dependent.

Although the hallmark of primary biliary cirrhosis (PBC) is the presence of anti-mitochondrial antibodies (AMA), a significant number of patients have anti-nuclear antibodies (ANA) directed primarily against two nuclear proteins, gp210 and sp100. In PBC, there are considerable data on the specificity of these anti-nuclear antibodies as well as suggestive evidence that antibodies to gp210 predict a poor outcome. However, a further understanding of the significance of these autoantibodies has been hampered by limitations in accessing human subjects in a preclinical or early asymptomatic stage. To overcome this limitation, we have taken advantage of transgenic mice with abrogated transforming growth factor-ß signalling in T cells (dnTGF-ßRII) that develop histological features of PBC as well as the same AMA specificity. We studied these mice for serum ANA, including specific autoantibodies against gp210 and sp100. We further examined sera from dnTGF-ßRII mice with concurrent deletions of the genes encoding interleukin (IL)-12p35, IL-12p40, IL-23p19, IL-17, IL-6, interferon (IFN)-γ or tumour necrosis factor (TNF)-α. Sera from all the dnTGF-ßRII mouse lines contained antibodies against gp210 and sp100. Of significance, mice with germline deletions of the genes encoding IL-12p40, IL-23p19, IL-17, IL-6 and TNF-α had significantly lower titres of anti-gp210 antibodies. These results provide a platform to dissect the mechanisms of gp210 and sp100 autoantibody production in dnTGF-ßRII mice as well as to study the possible role of ANA in the pathophysiology of PBC.

The role of natural killer (NK) and NK T cells in the loss of tolerance in murine primary biliary cirrhosis.

One of the major obstacles in dissecting the mechanism of pathology in human primary biliary cirrhosis (PBC) has been the absence of animal models. Our laboratory has focused on a model in which mice, following immunization with a xenobiotic chemical mimic of the immunodominant autoepitope of the E2 component of pyruvate dehydrogenase complex (PDC-E2), develop autoimmune cholangitis. In particular, following immunization with 2-octynoic acid (a synthetic chemical mimic of lipoic acid-lysine located within the inner domain of PDC-E2) coupled to bovine serum albumin (BSA), several strains of mice develop typical anti-mitochondrial autoantibodies and portal inflammation. The role of innate immune effector cells, such as natural killer (NK) cells and that NK T cells, was studied in this model based on the hypothesis that early events during immunization play an important role in the breakdown of tolerance. We report herein that, following in-vivo depletion of NK and NK T cells, there is a marked suppression of anti-mitochondrial autoantibodies and cytokine production from autoreactive T cells. However, there was no change in the clinical pathology of portal inflammation compared to controls. These data support the hypothesis that there are probably multiple steps in the natural history of PBC, including a role of NK and NK T cells in initiating the breakdown of tolerance. However, the data suggest that adaptive autoimmune effector mechanisms are required for the progression of clinical disease.

The multi-hit hypothesis of primary biliary cirrhosis: polyinosinic-polycytidylic acid (poly I:C) and murine autoimmune cholangitis.

A void in understanding primary biliary cirrhosis (PBC) is the absence of appropriate animal models. Our laboratory has studied a murine model of autoimmune cholangitis induced following immunization with 2-octynoic acid (2OA), an antigen identified following extensive quantitative structural activity relationship (QSAR) analysis, using human autoantibodies and three-dimensional analysis of the mitochondrial autoantigen, the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2). Mice immunized with 2OA coupled to bovine serum albumin (BSA) develop anti-mitochondrial antibodies (AMAs) of the identical specificity as humans with PBC, and in addition develop inflammatory portal cell infiltrates in liver. However, the natural history of disease is less severe than in humans and does not include fibrosis. Data from human and autoimmune murine models suggest that environmental and/or infectious agents can exacerbate autoimmune reactions, and a model of PBC has been described in which polyinosinic-polycytidylic acid (poly I:C), a viral RNA mimetic and Toll-like receptor 3 (TLR-3) agonist induces low-titre AMAs and in mild portal infiltrates. We took advantage of our established model to determine whether immunization with 2OA-BSA coupled with poly I:C alters the disease process. Indeed, the addition of poly I:C produces a profound exacerbation of autoimmune cholangitis, including a significant increase in CD8(+) infiltrating T cells, as well as a marked increase of proinflammatory cytokines. In addition, mice have evidence of fibrosis. These findings lend support to the concept that besides breakdown of self-tolerance, there is a requirement of a second 'hit' during the breakdown process that leads to disease which more faithfully mimics human PBC.

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.