Transforming growth factor-beta (TGF-β) has pleiotropic effects on cell growth and immunological control with a promoting effect on the development of the regulatory T-cell compartment [13]. Overexpression of a dominant negative form of the TGF-β promoter leads to development of PBC-like features with lymphoid cell infiltration of portal fields as well as colitis with 100% AMA positivity [14, 15]. The adoptive transfer of CD8+ cells from these animals into immunodeficient Rag1 ^−/− mice underlined the importance of CD8+ cells, since these mice developed similar histopathology to human PBC; however, CD4+ T-cell transfer had no effect on the liver phenotype but worsened colitis [16]. Further studies with an anti-CD20 antibody in young dnTGFβRII showed complete loss of serum AMA positivity and decreased liver inflammation, but were ineffective when initiated in mice with established disease [17]. A central role for natural killer T (NKT) cells in PBC pathogenesis is supported by the generation of CD1d^−/−-dnTGFβRII mice, in which reduced NKT function caused ameliorated inflammation, bile duct damage, mild ductopenia, cholestasis, and biliary fibrosis [18]. IL-12, consisting of a p40 and a p35 subunit, was studied by generating an IL-12p35 ^−/− and IL-12p40 ^−/− mouse strain on the dnTGFβRII background [19]. Whereas the IL-12p40 ^−/− mice were protected from liver inflammation, in IL-12p35 ^−/− mice, liver inflammation with similar severity but delayed onset compared to the parental dnTGFβRII mice was detected [19]. In addition, the deletion of IL-12p35 subunit from dnTGFβRII mice leads to frequent development of liver fibrosis with numerous immunological and histological features similar to human PBC [19]. To further characterize this interesting and promising mouse model, it will be crucial to study the effect of the different cytokines, including IL-12, -23, and -35 on liver phenotypes and on fibrotic changes via cytokine administration or cytokine-neutralizing antibodies [20].

Mice with genetic IL-2 receptor deficiency show 100% AMA positivity, lymphocytic portal inflammation, as well as CD4+ and CD8+ lymphocytes infiltrating the bile duct epithelium of intralobular bile ducts [21]. Interestingly, these animals show concomitant severe intestinal inflammation, which is usually not seen in PBC but PSC patients. No hepatic granuloma formation is seen in this mouse model [21]. In addition, there is no information on serum markers for cholestasis and whether also large bile ducts are involved.

Questioning the role of IL-12 in PBC triggered the generation of double knockouts via crossing IL-2Rα ^−/− and IL-12p40 ^−/− mice [22]. IL-2Rα ^−/− IL-12p40 ^−/− double-knockout mice show exacerbated autoimmune cholangitis, higher degree of liver fibrosis, and ameliorated colitis compared to IL-2Rα ^−/− single-knockout mice [22]. For more detailed characterization of cholestasis in this interesting mouse model, serum bile acid and alkaline phosphatase levels are awaited [22]. In addition, it would also be important to know whether IL-2Rα ^−/− IL-12p-40 ^−/− mice develop large duct disease.

The introgression of large genetic intervals on chromosomes 3 and 4 in nonobese diabetic (NOD) mouse strain leads to the development of NOD.c3c4 mice [23, 24]. On histological examination, in a high percentage, eosinophilic infiltration of bile ducts and autoreactivity against the PDC-E2 component are seen. To lower extend destructive cholangitis and granuloma formation can be observed. Whereas these animals show high seropositivity for AMA and ANA (80–90%), unfortunately, we do not have any information on cholestasis parameters of these animals. Intriguingly, extrahepatic bile duct disease is observed in NOD.c3c4 mice – a feature that would better fit to PSC rather than PBC – with development of cystic dilations of bile ducts, partial exfoliation of the biliary epithelium, and dense neutrophil-granulocytic infiltration [23]. The underlying mechanisms, however, for this peculiar phenotype is not clear and deserves detailed time-course studies (e.g., cholangiography or bile duct plastination for better characterization of large duct disease, characterization of the inflammatory infiltrate). The pronounced neutrophil-granulocytic infiltration of bile ducts could be, at least in part, a secondary phenomenon due to dilatation and secondary ascending cholangitis. Consequently bile culture studies should also be of interest. Interestingly however, treatment of NOD.c3c4 mice with a monoclonal antibody directed against CD3 protected these mice from cholangitis [23]. In general, due to the complex morphological changes in NOD.c3c4 mice, this mouse model may serve as a model for different cholangiopathies, including also several important aspects of PSC pathogenesis.

The observations that anion exchanger 2 (AE2) is downregulated in the liver and lymphocytes of PBC patients and that ursodeoxycholic acid restores AE2 expression and stimulates biliary bicarbonate secretion partially by activation of hepatic AE2 [25–27] were the trigger to generate Ae2a,b^−/− mice. This mouse model shares some immunologic and hepatobiliary features with PBC [28]. Histologically, mild to severe portal inflammation with high interindividual variations in regard to the liver phenotype is observed. In addition, the defective Treg cell function and CD8+ T-cell expansion seen in these mice could be due to the AE2 dysfunction, which seems to be critically involved also in the homeostasis of the immune system. However, so far a detailed characterization of this model in regard to investigation of large ducts and in regard to potential biliary fibrosis has been not performed. One major limitation of the model may lie within the fact that this mouse strain seems to be very difficult to breed (personal communication Juan Medina, Pamplona, Spain).

Scurfy mice with a selective loss of the transcription factor Fox-P3 (forkhead box P3, also known as scurfin) resulting in a functional deficiency of Treg cells show serological and morphological features of immune-mediated cholangitis, including severe bile duct injury [29, 30]. However, serum bile acid and alkaline phosphatase levels are not reported in these mice. Findings in scurfy mice underline the potential importance of Treg cells for the pathogenesis of PBC. One of the major limitations of this model is based on an extremely short life span of these mice of about 4 weeks, which seriously limits their use for longitudinal studies (e.g., disease progress, drug testing).

MRL/lpr mice with the lymphoproliferative gene lpr (also known as MRL/MP-lpr/lpr) spontaneously develop severe autoimmune-mediated disorders, such as vasculitis, glomerulonephritis, inflammation of salivary glands, interstitial pneumonia and plasma-cellular infiltration of portal fields with biliary injury, and development of AMA [31]. The relatively low percentage, about 50% of mice showing PBC-like features, critically limits the usefulness of these mice as a PBC model.

Currently no “ideal PBC model” exists among the available mouse models. Although an enormous progress has been achieved in the last decades in the generation of different model systems that show astonishing similarities with human PBC, concerning immunological and histological characteristics, each model harbors still its specific limitations. As PBC represents a chronic cholangiopathy with slow progression to biliary fibrosis and cirrhosis, long-term studies with detailed characterization of the cholestatic phenotype would be of major interest and urgent need for these models.

PSC leads to irregular scarring of the biliary tree causing bile duct strictures and dilatation-affecting intra- and extrahepatic bile ducts and may finally lead to biliary cirrhosis and liver failure. PSC primarily affects young men and is frequently associated with inflammatory bowel disease with specific clinical features including rectal sparing, right-sided disease, and backwash ileitis (i.e., PSC-IBD) [32]. The main attributes of an ideal PSC model therefore include the following clinicopathological features: male predominance, progressive fibrous-obliterative cholangitis of medium-sized and large bile ducts, onionskin-type-like periductal fibrosis, biliary type of liver fibrosis, concomitant predominantly right-sided mild colitis or pancolitis, and the high risk for CCA.

Animal models for (primary) sclerosing cholangitis [(P)SC] arbitrarily can be clustered into six different groups [33]: chemically induced cholangitis, knockout mouse models, cholangitis induced by infectious agents, models of experimental biliary obstruction, models involving enteric bacterial cell-wall components or colitis, and models of primary biliary epithelial and endothelial cell injury. Subtypes of models, their respective characteristics, and according references are summarized in Table 4.2. Due to limitations of space, we have to focus on only a few of them.

Bile ducts are major targets in acute and chronic GvHD representing a common complication and limiting factor of an allogeneic tissue and bone marrow transplantation. In humans, acute GvHD occurs within 100 days of transplant, and chronic GvHD (cGvHD) typically develops 100 days after transplantation. In mice, this temporal classification is not necessarily seen, since disease manifestation can differ in time of onset and is mainly defined by the clinical phenotype. Thus, chronic GvHD develops within weeks after transplantation in most mouse models [73]. Pathogenetically, cholangiocytes of small- to medium-caliber bile ducts are the major targets of T-cell-mediated destruction, causing apoptotic cell death and ultimately ductopenia [74]. So far, the detailed pathomechanism of GvHD is not clear.

In mice, GvHD across minor histocompatibility antigens can be induced experimentally by injection of spleen and bone marrow cells of congenic B10.D2 mice into sublethally irradiated BALB/c mice [60]. Bile ducts develop severe cholangitis with predominate lymphocytic inflammatory infiltrates 2–3 weeks after transplantation, and later on periductal fibrosis is observed. The major limitations of this mouse model are that neither loss of intrahepatic small bile ducts nor progression to liver cirrhosis during an observation period of 14 month is observed [60]. Generally speaking, factors confounding the translation of findings in mouse models to the human disease lie behind the fact that in humans acute GvHD typically precedes the chronic form, although in some cases chronic GvHD can occur without the occurrence of clinically obvious acute GvHD [73]. In addition, most patients are given immunosuppressive therapy to prevent acute GvHD influencing the development of chronic GvHD and further complicating modeling human GvHD in animals.

BA is the most frequent identifiable cause of neonatal cholestasis, and most patients require early liver transplantation [75]. To date, the underlying pathophysiological mechanisms are unknown, although a pivotal role for a dysregulation of cellular and humoral immunity, viral, toxic, and genetic factors are considered [75]. To date, different model systems for BA have been established, including young lambs and calves [76], sea lampreys [77, 78], zebrafish [79] and mice [79–83]. In newborn BALB/c mice, infection with rhesus rotavirus type A (RRV) in the first 2 days of life leads to liver disease with development of hepatobiliary injury and cholestasis within 1 week of infection [82, 83] mimicking human BA in several aspects [82–84]. Intriguingly, this mouse model shares major clinicopathological features with the human disease, including a time-restricted susceptibility of bile duct injury to the early postnatal period, acholic stools, bile duct proliferation, and portal inflammation as well as type 1 rich inflammatory infiltrate in the liver and bile ducts [84–90]. However, one of the main limitations of this mouse model is the high mortality rate of mice.

CCA is an epithelial biliary malignancy that originates from oncogenic transformation of cholangiocytes. Depending on the anatomic site, they may originate from different cell types, including intrahepatic biliary epithelial cells, hepatic progenitor cells, or mucin-producing cuboidal cholangiocytes of the extrahepatic biliary epithelium and peribiliary glands [91]. The identification of cellular origin in different subtypes may represent a prerequisite for effective therapy, but its impact on prognosis remains uncertain. CCA carcinogenesis is not entirely clear; however, well-known risk factors include the presence of PSC, liver fluke infections, hepatolithiasis or chronic hepatitis C, cirrhosis and toxins sharing induction of chronic cholestasis, and biliary and/or liver inflammation [92–95]. In the last years, several rodent models of CCA have been developed, including mice with xenograft and orthotopic tumors [96–102], genetically modified CCA models [103–105], and carcinogen-induced CCA models [106, 107]. Although these models provide adequate tools to gain insights into the pathophysiology of CCA development and to test new potential therapeutic agents in a preclinical setting, they harbor important limitations and difficulties discussed below (summarized in Table 4.3).