Genetic factors/susceptibility
Genetic factors have an impact on PBC pathogenesis that is stronger than that in nearly any other autoimmune disease [
85‐
87]. Accordingly, a concordance rate of about 60% was seen in monozygotic twins (as opposed to nearly 0% for dizygotic twins) [
88,
89]. A significantly increased incidence of PBC is seen in relatives of PBC patients [
89,
90]. The relative risk of a first-degree relative of a PBC patient is 50- to 100-fold higher than for the general population [
91], yielding a prevalence rate up to 5–6% [
19,
90,
92]. Interestingly, among affected monozygotic twins, though the age of disease onset is similar, progression and disease severity vary, emphasizing the role of epigenetic and probably environmental factors [
89].
It has been difficult to identify distinct susceptibility genes in PBC so far. Genetic associations in PBC were shown with major histocompatibility complex encoded genes. PBC is apparently associated with the DRB1*08 family of alleles, although marked variation is observed between different ethnic groups. Association with the DRB1*0801-containing haplotype is seen in populations of European origin, whereas in populations of Asian origin, an association is seen with the DRB1*0803 allele. A protective association has been described with DRB1*11 and DRB1*13, but once again, significant population differences are observed [
93‐
102]. Both associations of PBC with the DRB1*08 allele as well as the protective association of DRB1*11 and DRB1*13 were recently confirmed in the largest series ever reported including 664 unrelated patients from Italy [
103]. The odds ratio for developing PBC was 3.3 for DRB1*08-positive subjects, whereas it was reduced to 0.3 for subjects positive for DRB1*11, to 0.7 for DRB1*13, and to 0.1 for carriers of both DRB1*11 and DRB1*13. This study again highlighted the relevance of geographic variation, with marked differences in allele association between northern and southern Italy.
Associations have also been reported with polymorphisms of genes involved in innate or adaptive immunity. Allelic variations of tumor necrosis factor α (TNFα) and of cytotoxic T lymphocyte antigen 4 (CTLA-4), a key regulator of the adaptive immune system, have been repeatedly associated with susceptibility to different autoimmune diseases (such as type I diabetes mellitus and systemic lupus erythematosus) and were also associated with PBC [
104‐
109]. Although an association of CTLA-4 variants and susceptibility to PBC could not be demonstrated in all studies [
110,
111], Poupon et al. [
112] most recently confirmed a potential role of TNFα and CTLA-4 variants in the pathogenesis of PBC. In 258 PBC patients and two independent control groups of 286 and 269 healthy volunteers, the authors investigated distribution of newly identified htSNPs of 15 selected candidate genes: two related to immunity encoding for CTLA-4 and TNFα, ten genes related to bile formation encoding hepatobiliary transporters, and three related to adaptive response to cholestasis encoding nuclear receptors. In a case–control analysis, only haplotype-tagging single nucleotide polymorphisms (htSNPs) in CTLA-4 and TNFα showed differences in distribution between PBC and controls, confirming their potential role in the pathogenesis of PBC. In contrast, htSNPs of the ten transporter genes as well as the three nuclear receptor genes under study were equally distributed, confirming previous studies of htSNPs in key transporters without major impact [
1,
113,
114]. A strong association of the allelic variant TNFα rs 1799724 (C/T) with disease progression was shown. Most interestingly, a strong association with disease progression was also shown for AE2 rs 2303932 (T/A), a gene encoding for the apical anion exchanger 2 (AE2) in cholangiocytes and hepatocytes. In both cases, presence of the variant was associated with delayed disease progression. In a multi-variate Cox regression, the AE2 variant rs2303932 (T/A) was an independent prognostic factor for disease progression in PBC under UDCA treatment, in addition to serum bilirubin, alkaline phosphatase, and serum albumin levels which are established surrogate markers of prognosis in PBC [
112]. Most recently, a landmark genetic association study was published [
332]. Associations with the risk of disease were unravelled for 13 loci across the HLA class II region, two SNPs at the interleukin 12 alpha (IL12A) locus, one SNP at the interleukin 12 receptor beta 2 locus, and one previously described SNP at the CTLA4 locus. Associations with more than 10 further loci were described. Various associations with other loci have been described in individual populations, mostly of limited size. However, the vast majority have not been confirmed in independent cohorts, and to date, none of the genetic associations described in PBC have been proven sufficiently [
84,
88,
108].
Future genetic linkage studies in affected families as well as association studies in large cohorts of unrelated patients may disclose genetic variants conferring susceptibility or influencing progression and severity of disease. Such linkage studies are awaited for PBC [
115].
It remains speculative whether the female preponderance (gender ratio up to 10:1) reflects an X-chromosome-linked locus of susceptibility. Alternatively, a protective role of Y-linked genes could be assumed, or just a gender-specific exposure to environmental triggers like cosmetics [
116] or nail polishers [
117], as discussed below. However, speculation on a pathomechanistic role for X chromosomal genes was supported by the observation of an increased frequency of X chromosome monosomy in PBC as well as in other autoimmune diseases. This increase in X chromosome monosomy might lead to haploinsufficiency for specific X-linked genes and thereby increase disease predisposition [
118]. Case reports of PBC in patients with Turner syndrome (45, X0) also supported this hypothesis [
119].
Estrogen signaling has also been proposed to play a role in the homeostatic proliferative response of cholangiocytes in PBC. Accordingly, studies on polymorphisms in estrogen receptor genes revealed associations with the disease, at least in some populations [
120]. At the tissue level, cholangiocytes from PBC patients in the earliest disease stages (but not cholangiocytes from normal controls) express estrogen receptors [
121]. Agents able to modulate estrogen-receptor-mediated responses (such as tamoxifen) have therefore been proposed as novel, BEC homeostasis targeting therapies, and case reports support this hypothesis [
122,
123], but as yet, this potentially interesting therapeutic approach has not undergone formal assessment in clinical trials.
Most recently, altered expression of hepatic microRNA (miRNA) has been described in liver tissue of PBC patients [
124]. Certain miRNA negatively regulate protein coding gene expression and may play a critical role in various biological processes. However, a causal link between altered miRNA expression and the development of PBC still remains unproven.
Environmental factors
Despite strong evidence for a genetic background in PBC, epidemiological studies have early suggested a role for environmental factors in triggering and/or exacerbating PBC [
20,
125,
126]. A significant role for environmental factors was supported by the identification of geographic disease “hot spots,” as first reported in the northeast of England, using formal cluster analysis. The original UK analysis reported an increased frequency of PBC in former industrial and/or coal mining areas [
127]. Another recent study from New York examined the prevalence of PBC and PSC near superfund areas and reported significant clusters of PBC surrounding toxic sites [
128]. In synopsis, these observations gave rise to the hypothesis of a chemical environmental factor, potentially associated with contaminated land, which could either trigger disease or cause disease through a direct toxic effect [
84]. This hypothesis would also provide one possible explanation for the tissue tropism of PBC if the toxin or toxins are excreted into bile (and thereby concentrated in the biliary tree) [
84]. The observation that hormone replacement therapy and frequent use of nail polish are linked to the risk of developing PBC further supports the potential impact of environmental factors in the pathogenesis of PBC [
117]. Smoking also seems to be a risk factor for PBC and has been demonstrated to accelerate progression [
117,
129]. Associations of exposure to chemical environmental compounds and xenobiotics (including drugs, pesticides, or other organic molecules) with various human autoimmune diseases have been described as summarized in [
86].
Xenobiotics may contribute to the pathogenesis of PBC by triggering autoimmune reactions. Different mechanisms for the induction of autoimmunity by xenobiotics have been proposed [
86,
130]. A potential direct toxic effect of xenobiotics my cause cell death by apoptosis or oncosis, inducing the generation of immunogenic autoepitopes. In addition, chemical modification of native cellular proteins by removal and/or exchange of a hapten has been shown to change processing in antigen-presenting cells and may lead to the presentation of cryptic, potentially immunogenic peptides. Furthermore, xenobiotics may have the potential to modify host proteins to form neoantigens. Neoantigen-specific T cells and B cells, once primed, may cross-react with the formerly inert native autoantigens. In accordance with this hypothesis, Amano et al. [
131] studied a number of xenobiotics with a structure similar to lipoic acid, a residue on the E2 epitope of the pyruvate dehydrogenase complex (PDC-E2), the main autoreactive antigen identified in PBC so far in PBC. Replacement of lipoic acid by certain xenobiotics enhanced the reactivity of PBC sera against the PDC-E2 epitope. Particularly, one of the xenobiotics, 2-nonynoic acid, induced reactivity of PBC sera stronger than that of the native lipoic acid residue. Interestingly, the methyl ester of 2-nonynoic acid has a viol-/peach-like scent and is used as an ingredient in perfumes. It is ranked 2,324th out of 12,945 chemical compounds in terms of occupational exposure with an 80% female preponderance due to its use in cosmetics.
Infections
Among the environmental factors that have been suggested as potential causative agents in PBC, particularly different bacteria have been discussed. In early histologic lesions in PBC, non-caseating granulomas are observed, as seen in other granulomatous liver diseases including sarcoidosis [
1], drug reactions, and, most interestingly, infections. Furthermore, non-caseating granulomas are unique to PBC when compared to other autoimmune pathologies. This has led to suspicion of a microbial basis for PBC [
132]. In support of this hypothesis, certain bacteria were found to contain PDC components fully cross-reactive with the mammalian form. It was proposed that exposure to these homologues could trigger cross-reactive immunity. In favor of a bacterial etiology, recent data suggest that Toll-like receptor ligands induce an augmented inflammatory response in PBC. In combination, presence of cross-reactive antigens in a pro-inflammatory environment would theoretically be able to break tolerance [
133,
134].
With this theoretical background in mind, early studies associating various bacteria with PBC re-attract interest:
E. coli has been reported to be present in excess in the feces of patients with PBC. In addition, the incidence of urinary tract infections often induced by
E. coli is high in PBC patients [
73,
135], and history of urinary tract infections increases the risk of PBC [
117]. Another microorganism that has been proposed as a candidate for the induction of PBC is
Novosphingobium aromaticivorans [
136]. Titers of antibodies against lipoylated bacterial proteins of this ubiquitous organism, which metabolizes organic compounds including estrogens, were 1,000-fold higher compared to those against
E. coli in patients with PBC, but no antibodies were observed in a large cohort of healthy subjects.
Lactobacilli and
Chlamydia, which show some structural homology with the autoantigen (although reactivity against them is considerably less than that against either
E. coli or
N. aromaticivorans), have also been implicated as putative pathogens, as have
Helicobacter pylori and
Mycobacterium gordonae [
137‐
140]. Recently, a case of PBC following lactobacillus vaccination for recurrent vaginitis was reported. The vaccine contained
Lactobacillus salivarius, which exerts a high homology to the beta-galactosidase of
Lactobacillus delbrueckii (LACDE BGAL
266–280), and cross-reactivity of patients' autoantibodies against the human PDC-E2
212–226 epitope and LACDE BGAL
266–280 was found. Affinity to the
Lactobacillus epitope was higher than to the native mammalian, suggesting that antimicrobial reactivity may have preceded that to the self-mimic [
141]. However, the AMA status of this patient before repetitive lactobacillus vaccination could not be assessed and causal relation of lactobacillus exposure and development of PBC remains speculative also in this study.
Despite these intriguing associations, no compelling data have been provided to show that one individual infectious agent can reproducibly be detected in patients with PBC. Although attractive, the model of bacterial infections as cause of PBC is thus supported by little direct evidence. Further objective data are warranted, obtained either from prospectively followed cohorts or through case–control epidemiological approaches, confirming a role for bacteria in triggering PBC [
84].
An alternative infectious agent has recently been proposed as trigger of PBC when a human retrovirus was identified both in liver tissue and hilar lymph nodes from PBC patients. EM analysis of liver tissue obtained from PBC patients revealed retrovirus compatible particles in BECs. In periportal lymph nodes, mouse mammary tumor virus (MMTV) was detected and correlated with aberrant distribution of PDC-E2 in perisinusoidal cells. Homogenates of these periportal lymph nodes also had the capacity to infect BEC cultures inducing marked phenotypic change, and this effect could be abolished by irradiation of the culture media, suggestive of an infectious agent [
142,
143]. Retroviral infection hypothetically could cause BEC damage either through a direct viral cytopathic effect, through cross-reactivity between viral protein and self-PDC, a “molecular mimicry” model, or virus-induced apoptosis [
84]. Retroviral infection would also provide explanations for some key phenomena in PBC. PBC can recur rapidly after transplantation with all of the clinical manifestations including the detection of AMA in serum [
144], the aberrant expression of the AMA-reactive protein on BEC [
145], and histologic evidence of disease in up to 45% of patients [
146]. In this respect, the observation of an association of more potent immunosuppressive therapy following transplant and earlier and more aggressive recurrence of PBC [
147] is also of interest. MMTV replication is regulated in part by a progesterone-responsive glucocorticoid regulatory element in the promoter region, offering an alternative explanation for the female preponderance seen with PBC [
148].
These findings attracted attention in the field and were acknowledged by other investigators who pointed out the need for clinical trials with antiretroviral therapies [
149]. Subsequently, in a small non-randomized pilot study, therapy with Combivir (lamivudine + zidovudine) improved inflammatory scores, normalized AP, and reduced bile duct injury in patients with PBC [
150]. These findings await confirmation in a randomized, controlled trial.
Unfortunately, major findings of the outlined in vitro studies could not be reproduced by independent groups, and others raised concerns that these findings might mainly reflect contamination or technical artifacts [
151]. In an independent study, a large number of sera of PBC patients and healthy controls did not show reactivity against MMTV encoded protein, and no detectable immunohistochemical or molecular evidence for MMTV was found in liver specimens or peripheral blood lymphocytes [
152]. It was also speculated that beneficial effects of antiretroviral therapy could be partly explained by anti-apoptotic properties of nucleoside analogs [
151,
153]. Furthermore, mechanisms by which human betaretrovirus would enter human cholangiocytes are also not identified.
A more recent study, however, strengthened the case for involvement of retroviruses in (immune-mediated) liver disease: Sera of 179 patients with diverse chronic liver diseases and 31 controls were tested for reverse transcriptase activity and presence of human betaretrovirus by polymerase chain reaction. Reverse transcriptase activity was detected in 73% of autoimmune hepatitis patients, 42% of PBC subjects, 35% of patients with viral hepatitis, 22% of liver patients without viral or autoimmune pathogenesis (non-alcoholic fatty liver disease and alcoholic liver disease), and 7% of control subjects. In polymerase chain reactions, 24% of PBC samples were positive for human betaretrovirus compared to 13% in autoimmune hepatitis, 5% in other liver diseases, and 3% in non-liver disease control subjects [
154]. If these data can be confirmed, a retroviral compound in the pathogenesis of immune-mediated and viral liver disease seems attractive, though not specific for PBC. Thus, despite some intriguing findings, the pathogenetic relevance of retroviruses in the development of PBC remains enigmatic.