Mouse Models of Primary Biliary Cirrhosis

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Mouse Models of Primary Biliary Cirrhosis Axel R. Concepcion and Juan F. Medina* Division of Gene Therapy and Hepatology, CIMA, Clinic and School of Medicine University of Navarra, and Ciberehd, Pamplona, Spain Abstract: Primary biliary cirrhosis (PBC) is a chronic and progressive cholestatic liver disease of unknown etiopathogenesis that mainly affects middle-aged women. Patients show non-suppurative cholangitis with damage and destruction of small- and medium-sized intrahepatic bile ducts. Characteristically, the disease is strongly associated with autoimmune phenomena such as the appearance of serum antimitochondrial autoantibodies (AMA) and portal infiltrates with autoreactive T cells which recognize the inner lipoyl domain of the E2 component of the pyruvate dehydrogenase complex (PDC-E2). Here we review the major characteristics of a series of inducible and genetically modified animal models of PBC and analyze their similarities and differences with PBC features in humans.

Juan F. Medina

Keywords: Break of tolerance against PDC-E2, Antimitochondrial autoantibodies, Autoimmune cholangitis, Autoreactive T cells, Cytotoxic CD8+ T cells, Defective Treg cells, Cholestasis, AE2 deficiency, Deficient bicarbonate secretion. 1. INTRODUCTION PBC is a chronic and progressive cholestatic liver disease of unknown origin mostly affecting middle-aged women, in which an inflammatory process referred to as non-suppurative destructive cholangitis leads to injury and eventual loss of intrahepatic smalland medium-sized bile ducts [1-3]. Depending on the disease stage, portal and periportal inflammation may also be accompanied by important ductular reaction [4] and/or fibrosis. Cirrhosis and endstage liver disease may ultimately occur in untreated patients and in ~30% of patients who respond insufficiently to the current therapy of 13-15 mg/kg/day of ursodeoxycholic acid (UDCA) [5-7]. The disease is associated with autoimmune phenomena such as development of specific antimitochondrial autoantibodies (AMA) in the serum of 95% of patients [8] and coexisting autoimmune diseases in half of the patients, mainly Sjögren’s syndrome, Hashimoto’s thyroiditis, reumathoid arthritis, scleroderma and supervening autoimmune hepatitis (see also another review on mouse models for this process [9]) which in this context is characteristically known as the “overlap syndrome”. Additionally and regardless of AMA status, 30% of patients may develop different PBC-specific anti-nuclear autoantibodies (ANA) that present with multiple nuclear-dot or nuclear-membrane staining patterns [10]. These autoantibodies include the highly specific anti-gp210 and anti-p62 (with 95 % specificity for PBC each) as well as anti-sp100, anti-PML and antisp140 [1, 10]. However, the AMA developed in PBC patients are specifically directed against components of the 2-oxo dehydrogenase complexes, mainly the inner lipoyl domain in the E2 component of the pyruvate dehydrogenase complex (PDC-E2), but also the branched chain 2-oxo-acid dehydrogenase (BCOADC-E2) and 2-oxoglutarate dehydrogenase complex (OGDC-E2) [11]. Moreover, portal and periportal mononuclear infiltrates contain abundant autoreactive T lymphocytes against similar epitopes, particularly cytotoxic CD8+ T cells that surround the interlobular bile ducts and are mostly responsible for their injury [12]. Macrophages, eosinophils, and natural killer cells as well as plasma cells can also be present in the infiltrates. On the other hand, the regulatoy population of CD4+ T cells positive for the forkhead box protein 3 (FoxP3), i.e. the CD4+FoxP3+ Treg population, was reported to be under-represented *Address correspondence to this author at the Division of Gene Therapy and Hepatology, CIMA, Clinic and School of Medicine University of Navarra, and Ciberehd, Pamplona, Spain; E-mail: [email protected] 1381-6128/15 $58.00+.00

in the liver infiltrates (and peripheral blood) in patients with PBC as compared with appropriate controls [13]. Biochemical tests in PBC patients show that the serum levels of the cholestasis markers alkaline phosphatase (ALP) and -glutamyl transpeptidase (GGT) are commonly elevated, whereas an increase in serum levels of bilirubin is more restricted to patients with advanced stages of the disease. On the other hand, most patients have mildly elevated levels of serum aminotransferases (ALT, AST) and IgM, regardless their titer of AMA (>85% of which are of the IgG3 subclass) [14]. Experimental evidence supports the notion that bile-duct damage is initiated by environmental factors in genetically susceptible individuals [15, 16]. Recent genome-wide-association studies (GWAS) [17-19] and dense fine-mapping association studies [20] have related susceptibility to PBC with genetic variations in genes pertinent to immunity like HLA type II, IL12A and IL12RB2 genes. In Asian populations, additional studies using conventional genotyping reported no association of genetic single-nucleotide polymorphisms (SNPs) of the IL12-related pathway with PBC susceptibility [21]. In these populations differential PBC associations with SNPs of CTLA4 and SLC4A2/AE2 genes were found instead [22]. The identification and/or generation of genetically susceptible animal models of PBC (in mouse and/or other species) are contributing to unravel multiple pathophysiological aspects of this complex disease [1]. With respect to mouse models, Hayashi et al. [23] and Kanda et al. [24] reported over two decades ago, that a significant proportion of aged C57BL/6 mice may spontaneously develop PBC-like liver lesions and serum anti-PDC autoantibodies [23, 24]. While the report by Kanda et al. stressed that one of three aged female but not male C57BL/6 mice developed the PBC-like phenotype [24], Hayashi et al. found no gender-based differences [23]. More recently we could confirm that wild-type female C57BL/6 mice develop serum AMA (PBC-specific anti-PDC-E2), the titer of which increases with age (as determined by ELISA at 3, 6, and 12 months of age) [25]. In addition to mice with the C57BL/6 background, MRL/lpr mice were also found to spontaneously develop portal inflammation and cholangitis of small intrahepatic bile ducts [26], but failed to develop PBC-specific AMA against PDC-E2 [27]. Noticeably, MRL/lpr mice may exhibit autoimmune sialadenitis/Sjögren’s syndrome [28, 29], which in humans is frequently associated with PBC.

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Early in the second millennium there were also attempts to obtain animal models of PBC by sensitizing female mice of a range of strains (SJL/J, C57BL/6, NOD and BALB/c) with bovine PDC in complete Freund’s adjuvant [30]. Although each animal showed a specific antibody response following sensitization, development of non-suppurative destructive cholangitis during the experimental follow-up was found in the sensitized mice of the SJL/J strain only [30]. On the other hand, similar bile duct pathology could also be observed in this strain when casein was used instead of PDC for the breakdown experiments, indicating that the autoimmune cholangitis observed in this particularly susceptible SJL/J strain is antigen nonspecific [31]. The combination of genetic predisposition with triggering environmental factors is recognized as having a major role for the development of PBC in humans [32]. Therefore, a series of genetically modified animal models are being generated which, either spontaneously or in an inducible manner, can develop characteristic features of PBC. Though the available models frequently display additional features distinct from human PBC and none of them fulfill all criteria of such a complex disease, overall they are providing important mechanistic clues to understand the disease pathogenesis. Furthermore, these models may be useful when searching and testing alternative therapies for the disease. Our previous review in which we summarized the most relevant findings in spontaneous and inducible animal models in relation to major features of the disease in humans (cf. ref. [33] and Table 1 therein) is hereby updated and slightly expanded (see also recent reviews in [34-37]). 2. POLY I:C SENSITIZED MICE 2.1. Background Based on the previous observation that old C57BL/6 mice may spontaneously develop PBC-like characteristics [23, 24] Okada et al. designed an early development of PBC in this genetically susceptible mouse strain by administering polyinosinic: polycytidylic acid (usually abbreviated poly I:C). Poly I:C is a synthetic molecule structurally similar to double stranded RNA that functions as a viral RNA mimetic and toll-like receptor 3 (TLR-3) agonist and induces type-1 interferon (IFN) [38]. 2.2. Methodology and PBC Findings Female C57BL/6 mice were injected with 5 mg/kg of polyI:C twice a week for 28 consecutive weeks. Two months after the initiation of poly I:C injection, high numbers of infiltrating CD4+ and CD8+ T cells, macrophages and dendritic cells were detected in the liver, together with increasing levels of serum markers of cholestasis. After six weeks of poly I:C administration, almost 90% of C57BL/6 mice had developed PBC-specific AMA in their serum [38]. Remarkably, more recent studies using this mouse model revealed a decreased frequency of CD4+Foxp3+ regulatory T cells (Tregs) [39], which concurs with the findings in human patients with PBC [13]. 2.3. Other Considerations The aforementioned genetic predisposition of female C57BL/6 mice for developing PBC features could favor a gender bias in this model [24, 25]. On the other hand, the poly I:C sensitized mouse model does not develop liver fibrosis, that is frequently encountered in human PBC [39]. 3. MICE IMMUNIZED WITH 2-OCTYNOIC ACID-BOVINE SERUM ALBUMIN 3.1. Background The breakdown of self tolerance in PBC was hypothesized to have originated through molecular mimicry triggered by xenobiotic agents in genetically susceptible individuals. Molecular mimicry –

Concepcion and Medina

initially considered to be relevant for halothane hepatitis– was postulated between trifluoroacetyl (TFA)-protein adducts (arising from the metabolism of halothane) and the lipoic acid moiety of PDC-E2 [40, 41]. But the molecular nature of the interaction of anti-TFA antibodies and PBC-specific AMA against the inner lipoyl domain of PDC-E2 could be demonstrated to be clearly dissimilar [42]. After initial trials in rabbits using an additional battery of synthetic structures based on halogenated compounds –conjugated with the lysine residues of bovine serum albumin (BSA) to mimic the structural configuration of the lipoic acid moiety of PDC-E2 (see below and ref. [43])– an equivalent strategy was carried out in mice but using 2-octynoic acid (2OA). 2OA is a chemical xenobiotic frequently found in the environment, as it may be present in perfumes, lipstick, and many common food flavorings. This compound has the potential to bind lysine and replace the lipoyl domain of the immunodominant PDC-E2, leading to a chemical xenobiotic mimic, which may be sufficient to break self-tolerance [44]. 3.2. Methodology and PBC Findings Female C57BL/6J mice were immunized intraperitoneally with 2OA coupled to BSA (2OA-BSA; 100 μg/25μL) in complete Freund's adjuvant and subsequently boosted every 2 weeks with 2OABSA and incomplete Freund's adjuvant. Following a latency period within just a month, the immunized mice were shown to develop AMA and autoimmune cholangitis with a high number of liverinfiltrating CD8+ T cells and CD19+ B cells, as well as elevated serum levels of tumor necrosis factor (TNF- ) and IFN- [45]. 3.3. Other Considerations The natural history of the disease observed in 2OA-BSA mice is less severe than in humans and does not include fibrosis. Interestingly, however, co-immunization of 2OA-BSA mice with galactosylceramide ( -GalCer) –an invariant natural killer T (NKT) cell activator– led to the appearance of fibrosis in the liver [46]. On the other hand, co-administration of poly I:C during 2OA-BSA immunization of female C57BL/6J mice was recently shown to dramatically exacerbate the process of autoimmune cholangitis [47]. Moreover, these co-immunized mice exhibited marked increases in inflammatory cytokine production associated with an increase in effector CD8+ T cell infiltration of the liver. Moreover, poly I:C administration produced eosinophil infiltration and bridging fibrosis in the liver. These data further support the notion that, also in humans, natural environmental factors which are able to mimic the effect of poly I:C, may modulate the natural history of the human disease in PBC patients. 4. NOD.c3c4 MICE 4.1. Background The NOD.c3c4 congenic mouse model (also named NOD.B10 Idd9 B6 Idd3 Idd10) was developed by Wicker’s group from the parental strains NOD.B10 Idd9.1/9.2/9.3 and NOD.B6 Idd3/17/10/18 [48]. Unlike the NOD (non-obese diabetic) mice, which by 30 weeks of age have immune-mediated destruction of pancreatic cells and diabetes in 70–80% of the female animals [49], NOD.c3c4 mice are all effectively protected from autoimmune diabetes because of introgressed insulin-dependent diabetes (Idd) resistance alleles. 4.2. Methodology and PBC Findings A decade ago, Koarada et al. reported that these diabetesresistant NOD.c3c4 mice do spontaneously develop a biliary disease characterized by lymphocytic peribiliary infiltrates and progressive biliary obstruction, as well as hepatomegaly and autoantibodies in their serum [50]. Combination of Idd-resistant alleles in NOD.c3c4 mice was shown to be very important for the development of the different components of this disease model.

Mouse Models of Primary Biliary Cirrhosis

Current Pharmaceutical Design, 2015, Vol. 21, No. 00

Table 1. Mouse models of PBC. Model

Major PBC features

Other considerations

Referenced in:

Immune-base animal models Genetic predisposition of aged C57BL/6 mice

•

PBC-specific AMA

•

Portal infiltrates

Poly I:C injection in young female C57BL/6 mice

•

PBC-specific AMA

•

Portal infiltrates

•

Decrease number of Treg cells

•

PBC-specific AMA

•

Portal infiltrates with high number of CD8+ T cells and CD19+ B cells

•

Increased serum levels of TNFand IFN-

•

Female bias was reported

Section 1

Section 2

Co-immunization with -GalCer leads to fibrosis

Section 3

PBC-specific AMA in 50-60% of mice

-Sialoadenitis

Section 4

•

Portal infiltrates with CD4+ and CD8+ T cells and eosinophils

-Dilation of the common bile

•

Formation of granuloma

Infection of NOD.1101 mice with N. aromaticivorans

•

PBC-specific AMA

•

Infection of NOD.1101 mice with E.coli 2OA-BSA injection in NOD.1101 mice

2OA-BSA injection in young female C57BL/6 mice

NOD.c3c4 mice

dnTGF RII mice of the C57BL/6 background

–/–

IL-2R mice of the C57BL/6 background

Male scurfy mice (Treg-deficient Foxp3sf/Y; C57BL/6 background)

-No gender bias

Section 5.2

Portal infiltrates

NKT cells are responsive to glycosphingolipids of the cell wall of N. aromaticivorans

•

PBC-specific AMA

Damage is unrelated to NKT cells

Section 5.3

•

Severe biliary disease

•

PBC-specific AMA

Both male and female are used

Section 5.4

•

Portal infiltrates enriched with CD8+ T cells

•

Liver granulomas

•

PBC-specific AMA Portal infiltrates

-Inflammatory infiltration in the colon and lungs

Section 6

• •

Increased number of terminally differentiated KLRG1+-CD8 + T cells

•

Defective Treg cells

•

PBC-specific AMA

•


•

Increased serum levels of TNF- , IFN- , IL-6, IL-12p40 and IgA

•


•

PBC-specific AMA

•


•

Increased serum levels of TNF- , IFN- , IL-6, IL-12p40 and IL-18

•


-Upregulation of microRNA-21

-Old IL-2R –/– mice show autoimmune disorders like hemolytic anemia and inflammatory bowel disease

Section 7

-Deletion of CD8 leads to attenuated damage of the bile ducts but increased colon inflammation Section 8

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(Table 1) Contd…. Model

Major PBC features

Other considerations

Referenced in:

Ae2-deficient mice Ae2a,b–/– mice

•

PBC-specific AMA

•


•

Increased production of IFN- and IL-12

•

Decreased number of Treg cells

•

Spontaneous cholestasis

•

Spontaneous liver fibrosis

Constitutive defect of Ae2 in the whole body results in additional abnormalities

Section 9

Double knockout models Deletion of p40 and p35 in dnTGFbetaRII and IL-2R observed phenotype

–/–

mice (as well as IL-17A and others for the latter) results in modulation of the

Thus, 33-week old female NOD.c3 mice, i.e. NOD mice with the C57BL/6 Idd-resistance alleles (only on chromosome 3), showed lymphocytic peribiliary infiltration. About 95% of NOD.c3 mice were negative when tested for the production of antinuclear antibodies (ANA). On the other hand, NOD.c4 mice, i.e. NOD mice with the C57BL/10 Idd-resistance alleles (only on chromosome 4), exhibited production of ANA but not liver lymphocytic infiltration [50]. The autoimmune etiology of the spontaneously developed histopathology in NOD.c3c4 mice was confirmed by adoptive transfer of splenocytes in naïve NOD.c3c4 irradiated recipients [50]. Shortly afterward, it was reported that 50-60% of NOD.c3c4 mice can spontaneously develop the serologic hallmark of PBC, i.e. the PDC-E2 specific AMA, at the age of two months [51]. Moreover, the areas with affected biliary epithelium in the liver of NOD.c3c4 mice are infiltrated with CD4+ and CD8+ T cells, and eosinophils, which are seemingly leading to destructive cholangitis and granuloma formation as in human PBC [51]. Furthermore, NOD.c3c4 mice exhibit mild to severe inflammation of salivary glands (sialoadenitis) [52], a process that also in humans may lead to the Sjögren’s syndrome in a significant portion of patients with PBC.

Section 10

[53]. In contrast to NOD.c3c4 mice, NOD.1101 mice are only partially protected from having diabetes and do not spontaneously develop autoantibodies or liver alterations. Based on the genetic background, however, it was reasoned that this subcongenic strain might harbor some susceptibility genes for bile duct disease and serve as a suitable model to test microbial and xenobiotic triggering factors for developing PBC.

5. TRIGGERING FACTORS IN NOD-DERIVED MOUSE LINES

5.2. Novosphingobium aromaticivorans Infection 5.2.1. Background Among pathogens, the first candidate to test was N. aromaticivorans, a ubiquitous Gram-negative alphaproteobacterium that exhibits xenobiotic-metabolizing properties. Several proteins of this bacterium were found to have a high degree of homology with the dominant immunogenic lipoylated domain of PDC-E2, and may be lipoylated as well [54, 55]. Thus, molecular mimicry conferred by N. aromaticivorans could lead to PBC development, a hypothesis supported by previous findings in PBC patients showing an association with an antibody response to a particular group of alphaproteobacteria in these patients [54-56]. 5.2.2. Methodology and PBC Findings The aforementioned hypothesis was tested by infecting common mouse strains (C57BL/6, NOD, and SJL) with N. aromaticivorans, and indeed the infection was found to induce development of AMA together with liver lesions [57]. Particularly, the inoculation of N. aromaticivorans in NOD 1101 strain led to severe enlargement of the liver which exhibited chronic T cellmediated autoimmunity against small bile ducts like in PBC. Furthermore, liver disease was reproduced in immunodeficient mice upon adoptive transfer of conventional T cells from chronically diseased mice [57]. 5.2.3. Other Considerations Experiments carried out in CD1d-targeted C57BL/6 mice seemingly indicate that NKT cells responsive to unique glycosphingolipids of the N. aromaticivorans cell wall (presented in a context of CD1 molecules by antigen presenting cells) play an important role in the development of the biliary disease triggered by this alphaproteobacterium [57, 58]

5.1. Background NOD.1101 (NOD.B6 Idd10/18R2) is a subcongenic strain originated from NOD.c3c4 mice, in which the C57BL/6 chromosomal segment corresponding to the insulin-dependent diabetes resistance alleles Idd10 and Idd18 on chromosome 3 was restricted

5.3. Escherichia coli Infection Recent studies in which NOD.1101 mice were infected with E. coli resulted in a more severe biliary disease and higher AMA titer as compared with the N. aromaticivorans-infected NOD.1101 model. The aforementioned unique glycosphingolipids of N.

4.3. Other Considerations NOD.c3c4 mice may develop additional features which are not prototypical of PBC, i.e. dilation of the common bile duct and cholangiocyte proliferation [51]. In order to analyze the role of B cells for the alterations observed in these mice, Moritoki et al. developed a B-cell deficient Igμ–/– NOD.c3c4 mouse [52]. Noticeably, genetically B-cell deficient NOD.c3c4 mice showed a decreased number of non-B cells in the liver but similar degree of granuloma formation and bile duct damage as compared with B-cell sufficient NOD.c3c4 mice. Moreover, biliary cyst formation and sialadenitis were attenuated in B-cell deficient NOD.c3c4 mice, illustrating a critical role of B cells in exacerbating these abnormalities and in modulating specific organ pathology [52].


aromaticivorans are absent in E. coli, and therefore the immunopathology observed in the E. coli-infected NOD.1101 mice may be mainly dependent upon molecular mimicry [59]. 5.4. 2OA-BSA Immunization 5.4.1. Background The aforementioned results obtained in the genetically susceptible C57BL/6J mice upon immunization with 2OA-BSA [45] could also be reproduced in additional experiments using the NOD.1101 strain [60]. 5.4.2. Methodology and PBC Findings In this case, both female and male NOD.1101 mice (6-week old) were immunized by intraperitoneal injection of either 2OABSA or BSA alone (following a protocol as detailed above for 2OA-BSA immunization of female C57BL/6J mice). NOD.1101 mice immunized with 2OA-BSA, but not with BSA alone, developed high-titer serum AMA and histological features of cholangitis, including portal infiltrates enriched in CD8+ T cells and liver granulomas, similar to human PBC [60]. 5.4.3. Other Considerations Compared with the finding in the C57BL/6J strain, the portal infiltrates in the 2OA-BSA immunized NOD.1101 mice were closer to those observed in human PBC as they included granulocytes (which are rarely seen in portal infiltrates of 2OA-BSA immunized C57BL/6J mice) [60]. However, neither the eventual biliary fibrosis nor the characteristic female bias of human PBC could be observed in this model of 2OA-BSA immunized NOD.1101 mice. 6. TGF- RECEPTOR II DOMINANT-NEGATIVE MICE 6.1. Background TGF- is a cytokine with pleiotropic effects on cell proliferation, differentiation, migration, and survival that affects multiple biological processes, being able to act on virtually all cell types. TGF- has also been demonstrated to promote the development of peripheral CD4+FoxP3+ Tregs [61, 62], the regulatory T-cell population previously reported to be decreased in human PBC [13]. 6.2. Methodology and PBC Findings Because knockout mouse models for TGF- isoforms 1, 2 or 3 were each non-viable [63-67], a transgenic mouse model with a dominant-negative TGF- receptor in T cells was generated to assess the role of TGF- signaling in the regulation of these immune cells [68]. In this model, the TGF- receptor type II includes a truncated intracellular kinase domain and this dominantnegative form of TGF- receptor type II (dnTGF RII) is expressed under the control of the murine CD4 promoter (with no CD8 silencer to allow the expression of the transgene in both CD4+ and CD8+ T cells) [68]. When dnTGF RII binds any of the three isoforms of TGF- , it is incapable of mediating a signal transduction [68]. Interestingly, dnTGF RII mice were found to spontaneously develop non-organ specific autoimmune phenomena characterized by the presence of circulating autoimmune antibodies and inflammatory infiltration in the liver as well as colon and lungs [68]. Mild infiltration was also observed in stomach, duodenum, pancreas and kidney [68]. Further studies by Gershwin’s group on dnTGF RII mice of C57BL/6 background revealed several key phenotypic features of human PBC, including spontaneous production of AMA against PDC-E2, BCOADC-E2, and OGDC-E2 [69]. By weeks 22-24, 100% of dnTGF RII mice developed serum AMA, and at the age of 6-7 months the liver of these mice showed moderate to severe portal infiltrates of CD4+ and CD8+ T cells, NK, and B cells in association with bile duct damage, though no infiltrating eosinophils or granulomas could be observed [69]. Additionally, these transgenic mice showed increased serum levels of IFN- , TNF- ,


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IL-6, and IL-12p40 as well as IgA (but not IgM) [69]. Recent studies also suggest that the elevation of these pro-inflammatory cytokines in dnTGF RII mice could be related, at least in part, to upregulation of microRNA-21 in several tissues like liver, spleen, and colon [70]. The sequence of mature miR-21 is strongly conserved throughout evolution and possesses numerous potential targets, several of which may also play a role in the developed PBC-like phenotype. Altered expression of different microRNAs has also been reported in human PBC [71], though the status of human microRNA-21 in the disease remains to be specifically addressed yet. 6.3. Other Considerations TGF- is a key modulator of the FoxP3 [72]. Therefore, the breakdown of tolerance in the dnTGF RII mouse model is seemingly related to suboptimal suppressive ability of the CD4+CD25+FoxP3+ Treg cell population. In fact, recent experiments of adoptive transfer of CD8+ T cells from dnTGF RII mice to mice with targeted recombination-activating gene 1 (Rag1–/–) indicate that recipient Rag1–/– mice exhibit portal inflammation, and damage and loss of intralobular bile ducts [73]. A similar pattern is found when CD8+ T cells are cotransferred with CD4+FoxP3+ Treg cells from the same dnTGF RII mice [73]. However, when the adoptive transfer of CD8+ T cells from dnTGF RII mice is accompanied by Treg cells from wild-type C57BL/6 mice, less portal inflammation and decreased bile duct damage are observed in Rag1–/– recipients [73]. Interestingly, CD4+FoxP3+ Treg cells from C57BL/6 mice and those from dnTGF RII mice exhibited differential expression of Treg molecules like GARP (glycoprotein A repetitions predominant), CD73, CD101, CD103 and IL-10 [73]. Further bone-marrow chimera studies were carried out to assess whether the autoimmune cholangitis mediated by CD8+ T cells from dnTGF RII mice was cell intrinsic or extrinsic [74]. These studies support a “two hits” hypothesis suggesting that two sequential pathogenic events are important in this model: an increase in the number of terminally differentiated cytotoxic (KLRG1+)-CD8+ T cells and defective Treg cells [74]. This view may be translated to human PBC as it concurs with the aforementioned occurrence of autoreactive CD8+ T cells in portal infiltrates and selective defects in the circulating and liver-infiltrating Treg population described in PBC patients [13]. 7. INTERLEUKIN-2 RECEPTOR

(IL-2R /CD25)–/– MICE

7.1. Background Expression of the CD25 molecule, i.e. the chain of the IL-2 receptor (IL-2R ), occurs at specific stages in early T and B cell development and may be induced upon lymphocyte activation of T cells. CD4+FoxP3+ Treg cells may be in a constant state of activation by self antigens, and they generally express IL-2R as well. Mice with targeted IL-2R gene (IL-2R –/– mice) show normal development of T and B cells, but in their adulthood they exhibit progressive splenomegaly and lymph node enlargement due to polyclonal T and B cell expansion [75]. Old IL-2R –/– mice display autoimmune disorders, such as hemolytic anemia and inflammatory bowel disease [75]. 7.2. Methodology and PBC Findings More recent studies in IL-2R –/– mice (with a C57BL/6J background) indicate that they may also develop autoimmune portal tract pathology characterized by infiltration of CD4+ and CD8+ T cells and damage of bile ducts resembling human PBC [76]. In fact, IL-2R –/– mice aged 4-22 weeks developed serum AMA with reactivity against PDC-E2 (but not against BCOADC-E2 or OGDC-E2) [76]. The levels of both IgG- and IgA-AMA were significantly increased, while those of IgM-AMA remained unchanged [76]. Compared to control littermates, IL-2R –/– mice also showed increased serum levels of IFN- , TNF- , IL-2, IL-6,


IL-12p40 and IL-17A, and decreased levels of IL-4, IL-5, and IL-10 [76]. 7.3. Other Considerations The use of distinct double knockout models have revealed that the pathogenic mechanism leading to autoimmune cholangitis in IL2R –/– mice differs from the pathogenic mechanism involved in the inflammatory bowel disease that coexists in these mice [77]. Thus double knockout IL-2R –/–CD4–/– mice show increased biliary ductular damage and decreased inflammation in the colon, and do not develop AMA. By contrast, IL-2R –/–CD8–/– mice show serum AMA but attenuated damage of the bile ducts and increased colon inflammation [77]. These findings strongly support a major role of CD8+ T cells mediating biliary ductular damage in a context of Treg deficiency. These findings in IL-2R –/– mice together with the aforementioned Treg abnormalities observed in PBC patients strongly support the notion that Treg deficiency may play a key role in the breakdown of self tolerance in PBC. Such a notion is further supported by the observation of PBC features in a 5-year-old male child with IL-2R deficiency [78]. 8. FoxP3-DEFICIENT MICE (SCURFY) 8.1. Background FoxP3 is a critical regulator for the development of Tregs, and FoxP3 abnormalities have been reported to result in increased susceptibility to different autoimmune diseases in humans [79]. Scurfy is an X-linked recessive mouse mutant with defective Foxp3 gene which develops lymphoproliferative abnormalities. Hemizygous Foxp3sf/Y males show poor perinatal survival, most of them generally dying a few weeks after birth [80]. Thus adult fertile Foxp3sf/Y males are rarely obtained, and traditional breeding to generate Foxp3sf homozygous females is very difficult, forcing the researchers interested in carrying out experiments with the scurfy model to simply use Foxp3sf/Y males. 8.2. Methodology and PBC Findings The reported low frequency of Tregs in PBC patients [13] and the findings in the IL-2R –/– mice [76] encouraged researchers to study the Treg-deficient Foxp3sf/Y males generated through breading heterozygous female C57BL/6J.Cg-Foxp3sf/X with male C57BL/6J mice [81]. These scurfy male mice were found to exhibit serological, histological, and cytokine features characteristic of autoimmune cholangitis similar to human PBC [81]. At 3 to 4 weeks of age, 100% of these mice developed high-titer serum IgGAMA, IgA-AMA and IgM-AMA reactivities against PDC-E2. In addition, scurfy mice showed a moderate to severe lymphocytic infiltration accompanied by small bile duct damage and bile duct loss [81]. The majority of liver-infiltrating lymphocytes were CD8 + T cells, but there were also CD4+ T cells. Furthermore, dramatic increases in cytokines such as IFN- , TNF- , IL-6, IL-12p40, and IL-18 were found in the serum of these mice [81]. 8.3. Other Considerations Most probably due to difficulties in generating scurfy mice, no further characterizations have been carried out in this model. In any case, available data reinforce further the notion that functional defects of Tregs may constitute a major predisposing feature leading to autoimmune cholangitis, and indeed the FoxP3-deficient scurfy mice provide additional evidence linking Treg dysfunction to the loss of tolerance and development of PBC phenotype [81]. 9. AE2a,b-DEFICIENT MICE 9.1. Background Notwithstanding the notable occurrence of autoimmune phenomena in PBC, classical immunosuppressants are quite ineffective in this disease, while early therapy with UDCA, a bile


acid known to produce bicarbonate-rich hydrocholeresis, provides significant benefits in most PBC patients [5-7]. We therefore speculated that patients with PBC could have alterations in the biliary secretion of bicarbonate. This hypothesis was confirmed through PET analysis using 11C-labeled bicarbonate in untreated PBC patients, who failed to respond to the choleretic hormone secretin and did not increase biliary bicarbonate secretion [82]. Noticeably, the response to secretin was restored in these patients upon their treatment with UDCA for 3-6 months [82]. Biliary secretion of bicarbonate mainly occurs through electroneutral Na+-independent Cl–/HCO3– exchange across the plasma membrane of cholangiocytes mediated by the anionexchanger 2 (AE2) [83, 84]. In the human liver, AE2 is present at the lumenal membrane of bile duct epithelial cells (mainly in small and medium size bile ducts) and also in the canalicular membrane of hepatocytes, particularly those of the periportal area [85]. An apical location of the Ae2 exchanger in the hepatobiliary cells was also described in rat [86-88]. On the other hand, in situ hybridization studies in the human liver further indicated that the level of AE2 gene expression is much higher in cholangiocytes than in hepatocytes [89]. These data suggest that cholangiocytes are highly dependent on AE2 because of the major role of these epithelial cells for the secretion of bicarbonate to bile. In addition to the aforesaid findings, earlier studies in liver biopsies and peripheral mononuclear cells indicated that AE2 expression is diminished in PBC [82, 90]. Moreover, we found that bile duct cells isolated from PBC patients and cultured for a few passages exhibit abnormalities in the stimulated AE2 activity [91]. Though the aforementioned GWAS studies in Caucasian patients [17-19] did not observe an association of PBC susceptibility with SNPs of AE2/SLC4A2 gene, such an association was found in Japanese PBC patients [22]. On the other hand, SNP analyses across this gene in Caucasian patients with PBC revealed that two AE2/SLC4A2 variants may be influencing their AMA status [92], and that a synonymous variation in exon 6 can be associated with the progression of the disease [93]. 9.2. Ae2a,b-Knockout Methodology and PBC Findings To address the possible pathogenic role of AE2 abnormalities in PBC we generated a targeted mouse model in which the three major Ae2 isoforms, Ae2a, Aeb1 and Ae2b2 were deleted [94, 95]. Interestingly, Ae2a,b-deficient mice (of either FVB/N, 129/Sv, and/or BALB/c background) exhibited important characteristic features of PBC at both hepatobiliary and immunologic levels [35, 95, 96]. Thus over time most Ae2a,b–/– mice (but not their wild type littermates) developed serum AMA against PDC-E2. Additionally, Ae2a,b–/– mice eventually increased the serum levels of IgM and IgG. Moreover, progression of cholestasis was verified by increased serum levels of the hepatic isoform of alkaline phosphatase. Detailed studies of 15-month old Ae2a,b–/– mice revealed that nearly all animals developed splenomegaly. Moreover, splenocytes of these aged Ae2a,b–/– mice showed alkalinized intracellular pH (pHi), as one could expect for cells in which AE2-mediated acidification activity is relevant for their pHi homeostasis. Upon CD3 stimulation, Ae2a,b–/– splenocytes showed increased production of IFNand bioactive IL-12p70. CD8+ T cells showed net expansion which together with a maintained total number of CD4+ T cells, led to inversion of the CD4+/CD8+ ratio in Ae2a,b–/– mice. Curiously, however, the CD4+ subpopulation positive for CD25 and Foxp3, i.e. the frequency of Tregs was significantly diminished in the spleen and liver of Ae2a,b-deficient mice. Immunohistopathology of the liver showed that a significant proportion of Ae2a,b–/– mice had extensive portal inflammation rich in T lymphocytes (particularly CD8+ but also abundant CD4+) which surround damaged bile ducts. Finally, analysis of cholangiocytes isolated from Ae2a,b–/– mice showed a pattern of gene expression compatible with oxidative stress and increased antigen presentation (with particularly increased levels of the mRNA for the MHC class I molecule H2-D1). More recently, we found that AE2 is critical for CD8+ T cells to maintain pHi ho-


meostasis and modulate immune responses [97]. In the absence of AE2, CD8+ T cells were found to increase their pHi and to enhance their activation and cell proliferation in response to CD3 stimulation. Altogether, our data from the Ae2a, b–/– mice therefore enable us to suggest that Ae2 deficiency may lead to alterations in pHi homeostasis in immunocytes as well as altered gene expression patterns in cholangiocytes, resulting in hepatobiliary and immunological changes that reproduce those of PBC in humans. 9.3. Other Considerations Targeted disruption in our Ae2a,b–/– mouse model is constitutional rather than conditional and affects not only the liver and the immune system but the whole body. Therefore it was not surprising that this model exhibits other phenotypical alterations such as infertility in males due to blocked spermiogenesis and azoospermia [94]. Also, animals of both genders exhibit dysfunction of parietal cells in the stomach [98], and abnormalities in bones [99] and teeth [100102]. Nevertheless, the observed features resembling PBC point to Ae2a,b-deficient mice as a potentially useful animal model of the human disease. The role of decreased AE2 in PBC pathogenesis is further supported by our recent findings that miR-506 is up-regulated in cholangiocytes from PBC patients, binds the 3'UTR region of AE2 mRNA, and prevents protein translation, leading to diminished AE2 activity and impaired biliary secretory functions [103]. In view of the putative pathogenic role of decreased AE2 in PBC, miR-506 may therefore constitute a potential therapeutic target for this disease 10. STRATEGIES TO DISSECT MECHANISMS INVOLVED IN THE LIVER IMMUNOPATHOLOGY OF MOUSE MODELS A series of studies are currently being performed trying to dissect the mechanisms that lead to autoinmmune cholangitis in several of the aforementioned models, particularly in those affecting Treg cell functionality such as dnTGFbetaRII and IL-2R – /– mice. 10.1. Genetic Modifications in dnTGFbetaRII Strain Deletion of p40 (a heterodimeric subunit of IL-12 when it is paired with p35, but also of IL-23 if paired with p19) in genetically modified p40–/–-dnTGFbetaRII mice was found to prevent the liver alterations normally observed in the parental dnTGF RII mice [104]. This is in contrast to the outcome of the deletion of the other IL-12 heterodimeric subunit p35 (which is present in IL-35 in addition to IL-12). Thus p35–/–-dnTGFbetaRII mice were found to develop liver inflammation and bile duct damage with similar severity (though delayed onset) as the parental dnTGFbetaRII mice [105]. Noticeably, ~50% of these double targeted (p35–/–dnTGFbetaRII) mice exhibit liver fibrosis [105]. 10.2. Genetic Modifications in IL-2R –/– Strain In contrast to the findings in the genetically modified p40–/–dnTGFbetaRII mice, deletion of p40 in IL-2R –/– mice was found to exacerbate autoimmune cholangitis and promoted liver fibrosis, suggesting that, distinct from its canonical effects, p40 may be acting as a negative regulator of inflammation in the liver of the IL2R –/– mouse model [106]. In the colon, however, p40–/–-IL-2R –/– mice showed reduced colitis as compared with the parenthal IL2R –/– mice [106]. Since the heterodimeric subunit p40 may also pair with p19 to form IL-23 and IL-23 plays a critical function for Th1/Th17 cells [107], deletion of IL-17A was carried out in IL2R –/– mice. Interestingly, IL-17A deletion led to more severe autoimmune cholangitis but ameliorated colitis as compared with single knockout IL-2R –/– mice [108]. Moreover, the liver of double knockout IL-17A–/–IL-2R –/– mice exhibited a marked increase in


7

pathogenetic CD8+ T cells. On the other hand, deletion of IFN- in double knockout IFN- –/–-IL-2R –/– mice showed no impact on cholangitis or colitis as compared to single knockout IL-2R –/– mice [108]. Altogether, the these data suggest that IL-17A may play a protective role in autoimmune cholangitis, while it can play a proinflammatory role in inflammatory bowel disease [108]. 10.3. 2OA-BSA Immunization of Genetically Modified Strains The use of 2OA-BSA to immunize mice deleted of IL-12/IL23p40, IL-12/IL-35p35, IFN- , IL-23p19, IL-17A, IL-17F and IL22 had shown that whereas IFN- has a pivotal role in the early events involved in the pathogenesis of 2OA-BSA-induced autoimmune cholangitis, the IL-23/Th17 pathway potentiates the effects of IL-12/IFN- -mediated immunopathology [109]. 11. OTHER ANIMAL MODELS OF AUTOIMMUNE CHOLANGITIS 11.1. Faenza Rabbits In addition to mice, other species may also be useful as animal models of autoimmune cholangitis. Over three decades ago, a PBClike phenotype was reported in an inbred rabbit strain from Faenza (Italy), which spontaneously developed non-suppurative destructive cholangitis and serum antimitochondrial antibodies [110]. The etiology of this pathology still remains unknown and no additional in depth-studies in the Faenza rabbits were reported to indicate whether the encountered antibodies were PBC-specific AMA recognizing PDC-E2 epitopes. Authors speculated that the PBClike phenotype could have been triggered by non-aflatoxic contaminants the animals were receiving in their food in a context of a genetic predisposition [110]. 11.2. Immunization of Wild-Type Rabbits and Guinea Pigs 11.2.1. Background The hypothesis that the breach of self tolerance in PBC may have originated through molecular mimicry triggered by xenobiotic agents in genetically susceptible individuals was initially tested in New Zealand White rabbits [43]. A battery of synthetic structures that mimic the structural configuration of the lipoic acid moiety of PDC-E2 (aromatic CF3 compounds, halogenated aromatic compounds and halogenated straight chain hydrocarbons, each conjugated with the lysine residues of BSA) were employed [43]. A highly promising conjugate was then used in guinea pigs as well [111]. 11.2.2. Methodology and PBC Findings in Rabbit Female New Zealand White rabbits were divided into three groups and subcutaneously immunized, first with a mixture of compounds (100 μg per compound/animal) in the presence of complete Freund's adjuvant, and then boosted every 2 weeks with the compound mixture and incomplete Freund's adjuvant. Two compounds, 6-clorohexanoate-BSA and more in particular 6bromohexanoate (6BH)-BSA, were identified as being capable of inducing PBC-specific AMA in these animals [43]. The serum levels of alkaline phosphatase were also found to be significantly raised four months after 6BH-BSA sensitization. However, neither increased serum levels of transaminases and bilirubin, nor liver histological changes could be observed in the immunized rabbits [43]. 11.2.3. Methodology and PBC Findings in the Guinea Pig More recent experiments were carried out in female guinea pigs that were subcutaneously immunized with 30 μg of 6BH-BSA in complete Freund's adjuvant, and then boosted every 2 weeks with 6BH-BSA in incomplete Freund's adjuvant. In this species, 6BHBSA sensitization resulted in both AMA production and autoimmune cholangitis [111].


11.2.4. Other Considerations In addition to obvious genetic differences between rabbits and guinea pigs, species differences in biliary architecture and physiology (which in guinea pig might be closer to the human features), may have a role in the development of true PBC-like lesions in the latter animal model only [111]. 12. PROMISING CONTRIBUTION OF THE MOUSE MODELS OF AUTOIMMUNE CHOLANGITIS TO SEARCHING FOR THERAPEUTICS OF PBC In addition to a better understanding of the pathogenesis of the disease (Fig. 1A), an important objective of generating mouse models of autoimmune cholangitis is their subsequent use when searching for novel targets and additional therapeutic strategies for PBC in humans. In the case of Ae2a,b-knockout mice, however, the process has been rather the opposite: the efficiency of the only approved strategy currently available, i.e. a daily administration of middle-high doses of UDCA, pointed to defects in AE2-mediated bicarbonate transport as being potentially involved in PBC pathogenesis (Fig. 1B). In addition to UDCA, preliminary data of initial clinical trials with obeticholic acid (INT-747) suggest that FXR agonists may also be efficient in PBC [112], and experiments


in Mdr2–/– mice (Mdr2 is the mouse ortholog of the human gene MDR3/ABCB4) indicate that INT-767 (which is 100-fold more potent than INT-747 as FXR-agonist) increases bile flow and biliary bicarbonate secretion and reduces the liver injury [113]. With respect to the other animal models of autoimmune cholangitis mentioned above, the 2OA-BSA mouse model and dnTGF RII mice are being actively employed to identify novel potential therapeutic strategies for PBC that are more directly related to the immune system. Recent data from Tanaka et al. indicate that interference of the CD40/CD40-ligand (CD40L) interaction results in a non-sustained reduction of liver inflammation in dnTGF RII mice [114]. CD40/CD40L interaction is known to provide key signals between cells of the adaptive immune system and its inhibition might be therapeutically promising for patients with aggressive PBC. The inhibitory molecule CTLA-4 binds to CD80/CD86 thereby preventing CD80/86 from interacting with CD28 and subsequent full T-cell activation. Accordingly, the commercially available immunoglobulin CTLA-4-Ig BMS-188667 (a soluble recombinant human fusion protein comprised of the extracellular domain of human CTLA-4 linked to a modified portion of the Fc domain of human IgG-1) was recently used in the 2OA-BSA model to further

Fig. (1). Available animal models of PBC support two major pathogenic mechanisms for the breach of tolerance and development of autoimmunity. (A) Primary defects of immunoregulatory factors in genetically susceptible individuals facilitate that either bacterial molecules mimicking the PDC-E2 epitope, xenobiotically modified PDC-E2 or spillage of native mitochondrial autoantigens derived from apoptotic biliary epithelial cells (BECs) trigger autoimmunity. For this to occur the epitope is presented by APCs via MHC class II to autoreactive CD4+ T cells and consequently these cells will activate cytotoxic CD8+ T cells against BECs. Moreover, the activation of PDC-E2-specific B cells leads to their differentiation into plasma cells and production of AMA. The interaction between BEC apotopes, macrophages and AMA can result in potent production of injurious cytokines which can exacerbate apoptosis of neighboring cells and recruitment of immune cells. Defects of Treg cells can aggravate this series of events (B) The loss of tolerance against biliary epithelial cells in PBC could also be due to reduced expression of the AE2 exchanger in both biliary epithelial cells and lymphocytes in genetically susceptible individual. Thus, endogenous and/or environmental factors may be responsible for the decreased expression of AE2, which in BECs can result in oxidative stress and cell damage. Cell injury may also be magnified through ineffectiveness of an adequate “bicarbonate umbrella” and abnormalities in antigen presentation. In lymphocytes, AE2 deficiency may alter further CD8 homeostasis and decrease the number of Treg cells.


potentiate subsequent inhibitory signals [115]. Noticeably, all manifestations of cholangitis, including intrahepatic T-cell infiltrates and bile duct damage as well as development of AMA, were prevented in mice that had been pretreated with CTLA-4-Ig one day before administration of 2OA-BSA [115]. When CTLA-4-Ig was administered to 2OA-BSA mice which have an already established autoimmune cholangitis, less but significant therapeutic benefit was achieved, with reduced intrahepatic T-cell infiltrates and biliary cell damage, while AMA titer remained unchanged [115]. Regarding the potential use of anti-IL-17A therapy in patients with PBC, the data obtained in double knockout IL-17A–/–IL-2R –/– mice (cf. above) suggesting that IL-17A may play a protective role in autoimmune cholangitis, should be carefully considered in relation to the design of novel strategies [108]. And regarding immunotherapy of PBC the aforementioned beneficial effects achieved experimentally in Rag1–/– recipients when using fully competent Treg cells [73] sound potentially feasible and promising. Finally, the available animal models of autoimmune cholangitis should be envisioned as complementary rather than autocomprehensive and auto-exclusive. Advances in the understanding of the major homeostatic role of an adequate transport of bicarbonate for the physiology of lymphocytes and biliary cells might be therapeutically useful and tested in a series of immune-based PBC models. On the other hand, and taking into account that the pathogenic events are ultimately confluent for all models (Fig. 1), the novel immune-based therapies could also be tested in the Ae2deficient mouse model when designing efficient therapies for PBC that may improve further the benefit achieved with UDCA. 13. MOUSE MODELS OF PRIMARY SCLEROSING CHOLANGITIS Mdr2–/– mice were mentioned above when dealing with the beneficial effects of FXR-agonists on biliary pathology [113]. But Mdr2–/– mice are not considered as a model for PBC but rather a model for another biliary disease named primary sclerosing cholangitis (PSC). PSC is a chronic cholestatic disease of unknown etiology in which there is inflammation of intra- and extrahepatic bile ducts, associated with biliary fibrosis, duct strictures and proximal dilations. In contrast to PBC, PSC affects mainly men. It is frequently connected with inflammatory bowel disease (mainly the ulcerative colitis), and shows a marked risk of malignancies, particularly of the biliary tract and colon. No efficient pharmacological treatments are available for PSC and liver transplantation remains yet as the standard of care for patients with advanced disease. PSC is viewed as an inflammatory-mediated cholangiopathy while there is controversy as to whether it should be regarded as an autoimmune disease [116, 117]. Although patients may develop numerous autoantibodies, none of them are as disease specific for PSC as the anti-PDC-E2 autoantibodies are for PBC [116, 118]. Indeed PSC patients show a relatively high prevalence of atypical perinuclear antineutrophil cytoplasmic antibodies (pANCA) but the high sensitivity of these antibodies is contrasted by their low specificity and poor correlation with disease activity [119]. ANA and additional autoantibodies against smooth muscle antigen (SMA), endothelial cell (AECA), cardiolipin, thyroid peroxidase (TPO), and rheumatoid factor may also be found in PSC but, similarly to pANCA, they all possess low specificity [116]. In fact, they are frequently viewed as nonspecific epiphenomena of an immune response against dying cells at an inflammatory site (for instance, dying neutrophils, which may promote development of pANCA) [118, 120]. The abundant series of current mouse models of PSC had been recently reviewed by members of the International PSC Study Group (IPSCSG), either jointly or separately [36, 116, 121]. In summary, PSC mouse models may be classified within seven major groups: i) mice with cholangitis chemically induced by either 3, 5diethoxycarbonyl-1, 4-dihydrocollidine (DDC) or lithocholic acid;


9

ii) cholangitis in genetically modified mice: with a point mutation in the ferrochelatase gene (fch/fch mice), Mdr2–/–/Abcb4–/– mice, and Cftr–/– mice; iii) mice with cholangitis upon infection with either Cryptosporidium parvum or Helicobacter hepaticus; iv) C57BL/6J mice with experimental biliary obstruction; v) mice undergoing experimental cholangitis associated with colitis induced by dextrane sodium sulfate (DSS); vi) mice with experimental biliary epithelial and endothelial cell injury because of graft-versushost disease (GVHD); and vii) mice with antigen-driven biliary injury, particularly the OVA-BIL mice –a transgenic mouse model with a construct leading to aberrant biliary expression of a membrane form of ovalbumin (OVA) under an apical sodium-dependent bile acid transporter (ASBT) promoter region– which are then subjected to adoptive transfer of OVA-specific T cells [122]. The aforementioned reviews [36, 116] are encouraging investigators to better characterize the available models as true models for PSC. But attempts to view them as autoimmune disease models are actually hindered due to the fact that no disease-specific adaptive immune responses have yet been recognized in human PSC. 14. CONCLUSION There is a current blossoming of animal models of human PBC, which taking into account the increasing difficulty to access human tissues, may allow for progress in understanding the molecular pathogenesis of this complex and multifactorial disease and achieving highly efficient therapies. Each model has advantages and disadvantages in this regard. However, it is not surprising that even highly attractive genetically modified mouse models include in most instance additional features that differ from the human liverspecific disease due to a widespread blocking of important signaling pathways (e.g. TGF- signaling) or gene expression (e.g. IL2R , FoxP3, Ae2). Interestingly, several models agree on the loss of tolerance by a failure of Tregs (Fig. 1), although a complete abolition of this lymphocyte population triggers a generalized autoimmune phenotype that affects not only the liver but other tissues as well. Disease induction with pathogens and/or xenobiotics in some of the current genetically modified animal models may constitute a promising approach to pave the way for further progress not only in understanding the pathogenesis of PBC but also in the design of effective therapeutic tools, particularly for those patients who respond insufficiently to the few drugs currently available. CONFLICTS OF INTEREST The authors confirm that this article content has no conflicts of interest.

ACKNOWLEDGEMENTS This work was supported through the Spanish “UTE for CIMA project”, by grants from the Spanish Ministry of Science and Innovation (SAF2006-07818, SAF2009-11538 and SAF2012-35455), and by the Carlos III Institute of Health (Ciberehd CB06/04/0067). ARC had financial support through the Subprogram “Torres Quevedo” (Spanish Ministry of Science and Innovation & the European Union, PTQ-10-04247). LIST OF ABBREVIATIONS 2OA = 2-octynoic acid 6BH = 6-bromohexanoate -GalCer = -galactosylceramide AE2/ SLC4A2 = Cl–/HCO3– anion exchanger 2 /solute carrier family 4 member2 ALP = alkaline phosphatase ALT = alanine transaminase AMA = antimitochondrial autoantibodies


ANA ASBT AST BCOADC-E2 BEC BSA CTLA-4 DDC DSS ELISA FoxP3 GARP GGT GVHD GWAS HLA IFN Ig IL MDR3/ABCB4

= = = = = = = = = = = = = = = = = = = =

NK OGDC-E2 OVA pANCA

= = = =

PBC PDC-E2 pHi poly I: C PSC Rag SLCx SNPs TGF TLR TNFTPO Tregs UDCA

= = = = = = = = = = = = = =

anti-nuclear autoantibodies sodium-dependent bile acid transporter aspartate transaminase branched chain 2-oxo-acid dehydrogenase biliary epithelial cell bovine serum albumin cytotoxic T-lymphocyte-associated protein 4 3, 5-diethoxycarbonyl-1, 4-dihydrocollidine dextrane sodium sulphate enzyme-linked immunosorbent assay forkhead box protein 3 glycoprotein A repetitions predominant -glutamyltranspeptidase graft-versus-host disease genome-wide-association studies human leukocyte antigen interferon immunoglobulin interleukin multidrug resistance protein 3/ATP-binding cassette sub-family B member 4 natural killer 2-oxoglutarate dehydrogenase complex ovalbumin perinuclear antineutrophil cytoplasmic antibodies primary biliary cirrhosis pyruvate dehydrogenase complex intracellular pH polyinosinic: polycytidylic acid primary sclerosing cholangitis recombination-activating gene solute carrier family x single nucleotide polymorphisms transforming growth factor toll-like receptor tumor necrosis factor thyroid peroxidise regulatory T cells ursodeoxycholic acid

Concepcion and Medina [7] [8] [9] [10] [11] [12]

[13] [14]

[15] [16] [17] [18] [19]

[20] [21] [22]

[23] [24] [25]

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Received: January 27, 2015

Accepted: March 13, 2015

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