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Experimental evidence on the immunopathogenesis of primary biliary cirrhosis

Abstract

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease for which an autoimmune pathogenesis is supported by clinical and experimental data, including the presence of autoantibodies and autoreactive T cells. The etiology remains to be determined, yet data suggest that both a susceptible genetic background and unknown environmental factors determine disease onset. Multiple infectious and chemical candidates have been proposed to trigger the disease in a genetically susceptible host, mostly by molecular mimicry. Most recently, several murine models have been reported, including genetically determined models as well as models induced by immunization with xenobiotics and bacteria.

Introduction

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease of unknown etiology characterized by autoimmune-mediated destruction of small- and medium-sized intrahepatic bile ducts.1 Serologically, PBC is characterized by the presence of increased levels of immunoglobulin M (IgM), a high titer of serum antimitochondrial autoantibodies (AMAs) and, in some of patients, PBC-specific antinuclear antibodies (ANAs).1 From both a clinical and a pathogenetic point of view, PBC is considered a peculiar, yet representative, autoimmune disease.2 PBC preferentially affects women, with one of the highest female/male ratios (10:1) described in autoimmunity,3 and most cases present within the fifth and sixth decades of life, with only exceptional cases reported in teenagers. AMAs are present in about 95% of PBC cases, with a disease specificity close to 100%, and are therefore considered the serological hallmark of the disease. Histologically, PBC presents bile duct inflammation with consequent destruction/loss of intrahepatic bile ducts and development of fibrosis and biliary cirrhosis. There are four PBC histological stages: (i) portal tract inflammation with bile duct obliteration and granulomas; (ii) extension of inflammation to the periportal area; (iii) septal or bridging fibrosis, with ductopenia (over half of the visible interlobular bile ducts having vanished); and (iv) estab-lished cirrhosis virtually undistinguishable from end stage liver diseases of different etiologies. Epithelioid granulomas with no sign of caseous necrosis are the characteristic lesions of PBC and can be found at any stage around damaged bile ducts.4

The definitive diagnosis of PBC is made when all of the following three criteria are fulfilled: the presence of serum AMA, increased enzymes indicating cholestasis (i.e., alkaline phosphatase) for longer than 6 months and a compatible or diagnostic liver histology. A probable diagnosis is made when two of three criteria are present.

The most common symptoms include fatigue, which is present in about 19% of patients at diagnosis, pruritus in about 20% of patients at diagnosis, and jaundice,5 but because of the changing disease scenario jaundice is now a very rare sign at presentation.6

Although the complete pathways of PBC pathogenesis remain unknown, several clinical and experimental findings strongly support autoimmune mechanisms for bile duct damage.7 However, the question of what causes the disease remains unanswered.

The most accepted hypothesis states that PBC results from an environmental insult on a genetically susceptible background. In this scenario, adaptive, both humoral and cellular (CD4 and CD8 T cells), and innate immunity have been proposed as coplayers in immune-mediated liver damage.

This review includes a critical discussion of what is known and what we hope will soon be known regarding the causes of onset and perpetuation of liver damage in PBC.

In this regard, we will first discuss the somehow overlooked role of female predominance in autoimmunity in general and in PBC in particular. We will then review what is known of the genetic basis of PBC susceptibility and the contribution of environmental factors in its development. Third, we will illustrate the established evidence on the immunobiology of PBC with specific mention of the new line of research on innate immunity.

The sex ratio of autoimmune disease

Similar to Sjogren’s syndrome, systemic lupus erythematosus, autoimmune thyroid disease and scleroderma, PBC manifests the highest predominance in females, with over 80% of patients being women. To explain this sex bias observed in autoimmunity, three major working hypotheses have been investigated so far, i.e., the role of sex hormones, fetal microchimerism and X-chromosome defects.

Sex-associated hormones, e.g., estrogens, androgens and prolactin, which not only differ between males and females but also can vary according to age, have been the first candidates taken into account, mainly because of their ability to modulate immune responses. Estrogen-mediated modulation of the immune response can act at different levels, including regulation of lymphocyte homing to a target organ and antigen presentation, thus potentially influencing both organ specificity of autoimmunity and breakdown of tolerance. Estrogens are also capable of directly modulating both pro- and anti-inflammatory activities of CD4 T cells and are therefore capable of influencing the outcome of the CD4 T-cell-mediated immune response. Finally, sex hormones can have activational effects on the hypothalamic–pituitary–adrenal axis because of their ability to modulate stress responses. Based on these observations, it should be clear that sex hormones, particularly estrogens, may be central to the T helper 1/T helper 2 balance within sites of inflammation, thus determining an appropriate or inappropriate inflammatory response in infection, tolerance development or autoimmunity.8 In past decades, various studies have reported that PBC is associated with the occurrence of sex hormone dysfunction. Epidemiological studies have shown a presence of menstrual dysfunctions, a higher frequency of hysterectomy and a higher risk of osteoporosis, indirectly suggesting an active role of estrogens in the pathogenesis of disease. In a recent study, taking hormone replacement therapies following menopause was significantly associated with PBC, although this may be secondary to the proposed enhanced rate of bone loss in chronic cholestasis.9

Besides their immunological functions, estrogens could have direct effects on cholangiocytes, the specific target of immune-mediated damage in PBC. Cholangiocytes have indeed been reported to be able to express estrogen receptors. Estrogens could, therefore, directly target cholangiocytes through their receptors, inducing proliferation (also in response to damage) and/or secretory activities of cholangiocytes. It has been shown that cholangiocytes from patients with PBC, especially in the advanced histological stages, do not express estrogen receptors, thus suggesting a role of estrogen deficiency in the development of ductopenia in PBC. The possible influence of estrogens on damaging cholangiocytes in PBC still needs to be confirmed and further investigated.10

A second hypothesis on female predominance in autoimmune diseases is the persistence of fetal genome parts in women, a phenomenon known as fetal microchimerism. Persistence in the maternal circulation of fetal cells, even for decades after pregnancy, creates a microchimeric status in which fetal cells are semiallogeneic to the maternal immune system. Many studies in autoimmunity have tried to associate the presence of fetal microchimeric cells with an increased risk of developing autoimmunity in women. Microchimeric cells were indeed reported in peripheral blood mononuclear cells from patients with autoimmune diseases, such as scleroderma, thus suggesting that non-autologous cells might mediate a graft-versus-host disease-like reaction in these patients. Nevertheless, subsequent studies have failed to recapitulate these findings.11, 12 In PBC, most of the studies failed to find a significant difference in the frequency of male microchimerism in female PBC compared with controls.13, 14, 15, 16 We are convinced that fetal microchimerism may be involved in the pathogenesis of some autoimmune diseases, although available data are still controversial, but it does not seem to play a major role in the pathogenesis of PBC.

We have recently provided experimental data supporting a new fascinating hypothesis on the female predominance of autoimmunity based on major sex chromosome defects.17 This theory is based mainly on two observations: first, several genes that are key factors in the maintenance of immune functions and tolerance are located on the X chromosome and, second, diseases associated with constitutive X monosomy or its major structural abnormalities, such as Turner’s syndrome, are frequently associated with autoimmune features and in some cases with chronic cholestasis. Quite surprisingly, only a few studies have so far investigated the genetics of sex chromosomes in autoimmunity. X-chromosome inheritance displays a peculiar pattern compared with autosomal chromosomes, since women are functional mosaics for X-linked genes. In females, most genes on one X chromosome are silenced as a result of X-chromosome inactivation (XCI). The result of XCI is to achieve equivalent levels of X-linked gene products between males and females. More recent data have mined this dogmatic view by demonstrating that at least 15% of X-linked genes are capable of escaping XCI in healthy women and are thus expressed from both X chromosomes. Up to 10% of total X-linked genes manifest variable XCI patterns in different individuals.18 As a result of these observations, a role for the X chromosome was first proposed based on experimental evidence that women with autoimmune diseases have a significantly higher frequency of peripheral blood cells with a single X chromosome (i.e., X monosomy) compared with healthy women. Importantly, this was observed in diseases with different organ specificities, including PBC,19 scleroderma and autoimmune thyroid disease.20 Loss of an X chromosome is indeed preferential and more frequently involves a parentally inherited one,21 suggesting a possible critical involvement of X-chromosome gene product defects in the female preponderance of PBC and other autoimmune diseases, although new factors, such as microRNAs22 or epigenetics,23, 24 should not be overlooked. Other authors have suggested that women affected with specific female-preponderant autoimmune diseases, i.e., scleroderma, manifest a skewed XCI pattern in their peripheral white blood cells.25 In PBC, however, we failed to demonstrate such preferential inactivation.21

What causes the disease?

Genetics factors

A decisive role of genetic factors in conferring PBC susceptibility has been widely demonstrated, as in other autoimmune diseases.26 As with other autoimmune diseases, genetic components of PBC susceptibility are not related to a single gene and do not have a Mendelian inheritance. First insights into a genetic component come from early epidemiological studies. These studies reported that the risk of developing PBC was significantly increased if a first-degree relative suffered from the disease, thus suggesting some level of heritability. In general, previous data indicate that 1–6% of PBC cases27, 28 (Table 1) have at least one family member manifesting the disease and our most recent study on 1032 patients throughout the United States confirmed such data, showing that 6% of cases also had a first-degree relative affected.9

Table 1 Reported prevalence rates of PBC in families with one index case

A more recent study has also shown an increased incidence of AMA without any sign of disease in offspring and first-degree relatives of PBC patients. Such familial prevalence rates are significantly higher than general population prevalence estimates, thus indirectly indicating the existence of a genetic disposition.29 However, the difficulty and therefore a major limitation of such studies is that prevalence rates in the general population are still uncertain, and control groups are not always included in the family studies.

There is a more powerful tool that provides a means to estimate the genetic component in complex/multifactorial diseases, i.e., the comparison between disease incidence in monozygotic (identical) and dizygotic (non-identical) twins. In autoimmunity, the concordance rate in monozygotic twins for late-onset diseases has been shown to be, on average, well below 50%. We first reported that concordance rates for PBC are as high as 63% in eight monozygotic sets but null in dizygotic twins,30 thus strongly indicating a genetic contribution in PBC susceptibility. Of note, however, is the observation that in some concordant sets, the PBC phenotype as well as comorbidities varied significantly within one pair. We can hypothesize that the phenotype/genotype discordance could be due to different factors, including epigenetic factors (perhaps of the X chromosome, as suggested below),17 exposure to environmental factors or serendipity (see below).

The challenge to identify susceptibility gene(s) that predispose for the development of PBC is still open. The majority of such studies have not only been derived solely from case-control designs,31 but also were limited by poor-control-matching criteria and sample size or selection. A plethora of association studies have been conducted (reviewed elsewhere), mainly focused on immune genes that affect the immune system belonging to both the human leukocyte antigen (HLA) family and non-HLA immune modulator genes, including cytotoxic T-lymphocyte-associated antigen 4, interleukin (IL)-1, IL-10 and vitamin-D receptor. The discussion of these data goes beyond the aims of this article and details have been reviewed elsewhere.31, 32

Of interest, a more recent multicenter study reported the first genome-wide association study and identified IL-12 and its relative receptor as susceptibility genes for PBC.33 While we welcome the results of this enormous study, we also submit our concerns on the need to recapitulate these associations in independent (and possibly more homogeneous) cohorts of patients.

Environmental factors

Although it is almost certain that PBC susceptibility is conferred by an unknown genetic factor(s), gene alteration is not sufficient to trigger the disease. Exposure to certain environmental factor(s), even not harmful per se, could result in immune tolerance breakdown and are therefore necessary for PBC onset.

Two main environmental factors have been investigated in PBC, i.e., infectious agents (bacteria and viruses) and xenobiotics (chemical compounds).34, 35 Epidemiological and experimental evidence, as well as animal models, supports the key role of environmental factors and, in particular, xenobiotics in the development of PBC.

Infectious agents

A first insight on the role of infectious agents/chemicals as initial triggers of PBC comes from early epidemiological studies. Urinary tract infections have been reported by several authors to be more frequent in patients with PBC than in controls, with Escherichia coli being the main etiological agent.36, 37, 38 More recently, our 2005 epidemiological study on 1032 patients with PBC and 1041 rigorously matched controls9 confirmed the association of an increased risk of PBC not only with urinary tract infections but also with vaginal infections, lifestyle factors such as smoking, and previous pregnancies. We also showed that frequent use of nail polish was associated with a slightly increased risk of having PBC.

The most widely accepted mechanism by which infectious agents can trigger autoimmune responses in several autoimmune diseases is known as molecular mimicry.

The molecular mimicry hypothesis states that infectious agents contain short amino-acid sequences (epitopes) that share a sufficient degree of similarity with self-protein(s) and are therefore able to trigger a promiscuous immune response, both antibody- and cell-mediated. This aberrant immune response, in turn, can recognize both microbial and self-epitopes due to crossreactivity. In PBC, in which the main autoantigens are of mitochondrial origin, crossreactivity is not surprising due to the conserved sequence of mitochondrial enzymes across all species, from eubacteria to mammals. It has been proposed that mitochondria originated following uptake of bacteria into the precursors of eukaryotic cells and maintenance as intracellular symbionts. Thus, it becomes difficult to tease out a causal role for microbial proteins in pathogenesis given their phylogenetic relationship to the human autoantigen. One line of argument that we have taken is that the breakdown of immune tolerance with consequent induction of autoimmunity would be more likely to occur when the microbial protein is extremely similar in sequence and that it would not be necessary for tolerance breakdown to take place in the disease target organ. In this scenario, T-cell activation produces crossreacting T cells leading to self-tissue destruction, which ultimately perpetuates the autoimmune injury, possibly through the degeneracy of T-cell receptors and cross-priming. Several bacterial strains in addition to E. coli have been suggested as crossreactive agents in PBC, including Klebsiella pneumoniae, Proteus mirabilis, Staphylococcus aureus, Salmonella minnesota, Mycobacterium gordonae, Neisseria meningitidis and Trypanosoma brucei, and the yeast Saccharomyces cerevisiae has also been investigated in PBC with nonspecific results.39 Conflicting evidence has been obtained on the role of Chlamydia pneumoniae in the pathogenesis of PBC since its antigens were first detected in 100% of PBC explanted liver sections compared with 8.5% of controls, but this was not independently recapitulated.40, 41

Recently, our group has provided serological and molecular data suggesting that a ubiquitous xenobiotic-metabolizing Gram-negative bacterium, Novosphingobium aromaticivorans, is the best candidate thus far for the induction of PBC. Two main characteristics of this bacterium render it an ideal candidate for the induction of PBC: (i) it contains two proteins sharing amino-acid sequences with the immunodominant epitope of pyruvate dehydrogenase complex E2 (PDC-E2), with the highest degree of homology ever described; and (ii) it can metabolize organic compounds and estrogens. We reported not only that is N. aromaticivorans present in human fecal specimens (25%), but also that it is able to elicit a specific antibody reaction (up to 1000-fold higher than against E. coli) in patients with PBC, but not in controls.42 Most recently, Mattner et al. supported the pathogenic role of N. aromaticivorans being able to induce serum autoantibodies and PBC-like liver lesions in mice following immunization.43

A novel human beta-retrovirus has been recently reported in perihepatic lymph nodes and other samples from patients with PBC, thus suggesting its possible role in the pathogenesis of PBC.44 However, in an independent experimental study conducted with a more comprehensive molecular and immunological approach on a larger series of patients and controls, we could not confirm such a hypothesis.45 More recently, human beta-retrovirus has been found by real-time reverse transcriptase polymerase chain reaction in the plasma of patients with different liver diseases, including PBC, autoimmune hepatitis and viral hepatitis.46 Although these data have not yet been confirmed, they suggest the involvement of human beta-retrovirus in the pathogenesis of etiologically different liver diseases and are therefore not specific for PBC. We therefore strongly discourage the use of antiretroviral drugs eventually proposed as PBC treatment.47

Xenobiotics

Xenobiotics are understood as substances that are foreign to the biological human system. The mechanism through which these compounds could induce an aberrant immune response to self-proteins is based on the fact that they are believed to alter or to complex either to self- or non-self-proteins, changing their molecular structures. The altered protein could therefore induce an immune response. As is the case for molecular mimicry, such an immune response may in turn lead to cross-recognition of the self form, ultimately ending in immune tolerance breakdown and chronic autoimmunity.

Important evidence indicating a possible pathogenic role of an organic compound in PBC comes from recent experimental data obtained in our laboratory. Long et al. have demonstrated that specific halogenated organic compounds, once attached to the major mitochondrial epitope backbone, were able to elicit AMA production by sera from patients with PBC. Antibodies against such a modified mitochondrial epitope had a higher affinity than antibodies to the native form.48 Later, using a multiplex approach, a subsequent study determined that 2-nonynoic acid is recognized by antibodies that do not crossreact with the PDC-E2 native form present in PBC sera with high affinity. This is particularly interesting since this compound does not occur naturally and is found in several cosmetic products, including nail polish.9

Regulatory T cells

CD4+ CD25high regulatory T cells (Tregs) are a subset of T cells that are able to suppress inappropriate immune responses or regulate the cellular immune response.49 In this scenario, a quantitative or qualitative impairment in Tregs could represent an important key factor in the breakdown of self-tolerance.50 An important role of Tregs in triggering aberrant immune responses against self has been demonstrated not only in autoimmunity in general but also in liver autoimmune diseases such as chronic autoimmune hepatitis.51 Moreover, animal studies have demonstrated that transfer of T cells lacking the CD4+ CD25high Treg subset into athymic nude mice results in the development of various T-cell-mediated autoimmune diseases.52

In PBC, recent experimental data reported significantly lower frequencies of CD4+ CD25high Tregs as percentages of total T-cell antigen receptor-αβ+/CD4+ T cells in PBC patients compared with controls, a fact that may contribute to the immune tolerance breakdown in the disease.53 The possible role in the loss of immune tolerance of such cells49 is also supported by PBC animal models (see below).

What causes liver damage?

Immunobiology

The assumption that PBC is an autoimmune disease is supported by several lines of clinical and experimental evidence that make this condition somehow a model and a paradox for autoimmunity.54, 55 In fact, PBC shares features commonly found in autoimmunity, such as female predominance, multifactorial genetic predisposition, the presence of autoreactive T cells (CD4 and CD8) and the presence of disease-specific autoantibodies, i.e., AMAs and PBC-specific ANAs (Table 2). Such autoantibodies, however, represent the basis for PBC being a paradox, as their direct pathogenetic role is still poorly defined.55 Serum autoantibodies are detected in approximately 90% of patients, yet seronegative cases manifest similar histological features and disease progression.56 Further, in the autoimmunity paradigm, the passive transfer of autoantibodies should reproduce disease-specific clinical features, and experimental immunization with disease-specific antigens should reproduce a model disease. These aspects have been only partially reproduced in PBC.

Table 2 Summary of the autoantibodies detected in PBC sera using indirect immunofluorescence (IIF), immunoblotting (IB) or other serological methods

Moreover, in autoimmune diseases, the reduction of disease-specific autoantibody titers correlates with disease amelioration; this criterion is also poorly met in PBC, in which there is no correlation between pattern or titer of AMA and progression or severity of disease.57 Finally, most autoimmune diseases are responsive to immunosuppressive therapy, while no such agent has proven effective for PBC.58

However, the breakdown of immune tolerance seems crucial to PBC initiation,59, 60 possibly secondary to the unique apoptotic features of bile duct cells61, 62, 63 and the unique tolerogenic liver function.64, 65

Autoantibodies

Circulating AMAs are highly specific for PBC and are detected in nearly 100% of patients when tested for using techniques based on recombinant mitochondrial antigens (immunoblotting or enzyme-linked immunosorbent assay). The high sensitivity and specificity of AMAs make them one of the most specific diagnostic tests of human immunopathology,66 different from other autoimmune conditions,67, 68 including autoimmune hepatitis.69

In most clinical settings for initial screening, AMAs are usually detected by indirect immunofluorescence using rodent liver, kidney and stomach sections as a substrate, leading in some cases to false positive or negative results.

AMA specifically recognizes lipoylated domains within components of the 2-oxoacid dehydrogenase family of enzymes within the mitochondrial respiratory chain. Among its constituents, the major autoantigen recognized is the E2 subunit of PDC-E2. Less frequent autoantigens are the E2 components of 2-oxoglutarate dehydrogenase and branched-chain 2-oxoacid dehydrogenase complexes and the E3 binding protein.70, 71 All immunodominant epitopes contain a aspartic acid - lysine - alanine motif, with lipoic acid attached to lysine (K), which is necessary and/or sufficient for antigen recognition.72

Highly PBC-specific serum ANAs have been detected in as many as 50% of patients and are named after their immunofluorescence pattern, i.e., multiple nuclear dots and rim-like pattern. Multiple nuclear dots reactivity is based on the recognition of Sp100 and promyelocytic leukemia proteins (possibly also crossreacting with small ubiquitin-like modifiers).73 Rim-like reactivity reacts against proteins of the nuclear pore complexes (NPCs) that include gp210 (a 210-kDa transmembrane glycoprotein involved in the attachment of NPC constituents within the nuclear membrane), nucleoporin p62 (a glycoprotein located in the core of NPCs) and the inner nuclear membrane protein lamin B receptor. Serum anti-gp210 antibodies are detected in about 25% of patients with PBC (range: 10–40%), while anti-p62 and anti-lamin B receptor antibodies are found in about 13 and 1%, respectively. Unlike AMA, cross-sectional and longitudinal data demonstrated an association between the presence of PBC-specific ANAs, especially anti-NPC antibodies, and a poorer disease prognosis. The pathogenic role of these antibodies, however, has been poorly investigated so far and remains unknown.

Autoreactive T cells

T helper (CD4+) T-cell antigen receptor-αβ+ and CD8+ T cells are most commonly seen in portal tracts, in particular around damaged bile ducts in the liver tissue of PBC patients, strongly suggesting the involvement of cellular immune mechanisms in biliary damage,74, 75, 76, 77, 78, 79, 80, 81 as suggested by liver tolerance.82 In past decades, the nature and the role of cellular adaptive immune responses in PBC have been extensively characterized, showing that both CD4 and CD8 T cells participate in liver tissue damage. Autoreactive CD4 T cells specifically targeting PDC-E2 self-antigen have been reported both in the peripheral blood and in the liver of PBC patients, but not in controls. There is a specific 100- to 150-fold increase in the number of PDC-E2-specific CD4 T cells in the hilar lymph nodes and liver when compared with peripheral blood of patients with PBC, thus further supporting their role in liver damage. Shimoda et al. characterized their antigen specificity, showing that in HLA DR4*0101-positive PBC patients, a single epitope, i.e., a 163–176 amino-acid sequence that encompasses the lipoic acid-binding residue of the inner lipoyl domain of PDC-E2, is the immunodominant epitope recognized by autoreactive CD4 T cells. Functionally, these cells in PBC patients, but not in controls, are of a proinflammatory nature, as demonstrated by their ability to produce proinflammatory cytokines, such as interferon-γ.76

Autoreactive CD8 T cells have also been well characterized in PBC and are currently considered the major effectors of adaptive immunity in tissue injury encountered in PBC. The HLA class I restricted epitope for CD8 T cells, namely, the 159–167 amino-acid sequence, maps in close vicinity to the epitopes recognized by CD4 T cells, as well as by AMA. It is of note that the autoepitope for T cells, both CD4 and CD8 T cells, overlaps with the B cell (AMA) counterpart and includes the lipoylated amino acid of the inner lipoylated domain. Similar to CD4 autoreactive T cells, the recent use of tetramer technology has shown a 10-fold higher frequency of PDC-E2159–167-specific CD8 T cells within the liver compared with the peripheral blood of patients with PBC. Moreover, the precursor frequency of PDC-E2-specific autoreactive CD8 T cells is significantly higher in the early rather than late stage of the disease. Functionally, autoreactive CD8 T cells in PBC have specific cytotoxicity against PDC-E2 antigen as well as the ability to produce interferon-γ rather than IL-4/IL-10 cytokines,83 while IL-17 has been recently suggested to play a role in PBC.84

Innate immunity

The adaptive immune system recognizes and responds to antigens via highly specific T-cell receptors, while innate immunity recognizes distinct evolutionarily conserved structures usually shared by invading pathogens, termed pathogen-associated molecular patterns (PAMPs), allowing a high efficiency for rapid recognition and elimination of viruses, bacteria and fungi. PAMPs engage pattern-recognition receptors, such as Toll-like receptors (TLRs), expressed on cells of the innate immune system, i.e., monocytes, dendritic cells and natural killer cells, all of which are therefore able to modulate the function of adaptive humoral and cellular immunity.85 Only recently has the study of innate immunity as a potential activator of autoimmune responses received a significant impetus and is no longer overlooked by clinical immunologists.85 The liver has been recognized both structurally and functionally as a major organ of innate immunity. One of the largest resident populations of cellular components of the innate immune system, including natural killer and natural killer T (NKT) cells, resides in the liver. Functionally, the liver constantly expresses effective immune responses against a wide range of pathogens from viruses to multicellular parasites. In that sense, the ambience of an inflammatory milieu seems to be critical for effective immunogenic signals to be delivered to intrahepatic T cells. Mounting evidence shows that innate immunity likely contributes to the initiation and/or progression of liver damage. PBC exhibits specific immunological features, such as the presence of epithelioid granulomas, elevated levels of polyclonal IgM, hyper-responsiveness to CpG oligodeoxynucleotides, increased levels of natural killer cells and cytokine responses, which strongly suggest a crucial role of innate immunity in the pathogenesis of PBC (Table 3).

Table 3 Synopsis of recent findings from the study of innate immunity changes in PBC

IgMs are commonly elevated in PBC, independent of AMA or ANA status,2 and their reduction is usually observed during treatment.86 Our group reported that polyclonal hyperIgM is secondary to a chronic polyclonal innate immune response of memory B cells to specific bacterial PAMPs, such as unmethylated CpG motifs.87 In this study, following stimulation with synthetic oligodeoxynucleotides containing CpG motifs, CD27+ memory B cells in cultured peripheral blood mononuclear cells from patients with PBC secreted significantly higher amounts of polyclonal IgM compared with controls. In a subsequent study, our group reported that B cells in peripheral blood mononuclear cells exposed to CpG motifs express increased amounts of TRL9 and CD86 and greatly enhance the production of AMAs. This evidence strongly suggests a profound disease-specific dysregulation of B cells and supports the proposed link between bacteria and PBC pathogenesis. In this scenario, B cells are hyper-responsive to innate stimuli, such as microbial CpG motifs, and therefore contribute to the perpetuation of the autoimmune process.88

Monocytes have also been implicated in the pathogenesis of PBC, with their proinflammatory activity greatly enhanced in PBC. Functionally, once activated by PAMPs through TLRs, monocytes are able to release proinflammatory cytokines, including IL-1, IL-6, IL-18, IL-12 and tumor-necrosis factor-α, which are able to amplify adaptive T-cell-mediated immune responses against pathogens. Our data have shown that peripheral monocytes from patients with PBC challenged with different ligands for TLR2, TLR3, TLR4, TLR5 and TLR9 produce a significantly increased level of all proinflammatory cytokines compared with healthy controls.89 From the innate immunity perspective, these findings suggest that peripheral monocytes from patients with PBC are more sensitive to infectious stimuli, resulting in the secretion of proinflammatory cytokines. The mechanisms for such increased sensitivity are currently unknown but might reflect or be secondary to the higher frequency of recurrent Gram-negative bacterial infections (e.g., urinary tract infections) in PBC.

Therefore, both B cells and monocytes constantly exposed to bacterially derived products from portal blood could participate in the modulation of the adaptive cellular immune response and possibly also in its priming.

The role of NKT cells in autoimmunity is also attracting growing attention.90 NKT cells are innate effector cells, which are regulated by self- and non-self-glycolipid antigens presented by the antigen-presenting molecule CD1d. This activation allows a rapid NKT cell production of effector cytokines and chemokines, thus modulating both innate and adaptive immune responses. The first evidence of a possible involvement of NKT cells in the pathogenesis of PBC comes from our study reporting a higher frequency of CD1d-restricted NKT cells in PBC patients compared with healthy individuals and, as was the case for autoreactive T cells, CD1d-restricted NKTs were more frequent in the liver compared with peripheral blood in PBC patients.81 Chuang et al. more recently confirmed the increased number of CD1d-restricted NKT cells in the liver of a dominant-negative transforming growth factor receptor beta II (dnTGFβRII) mice, our PBC mouse model, compared with controls. They further investigated the function of such CD1d-restricted NKTs, showing increased interferon-γ production in hepatic CD1d-restricted NKTs after exposure to α-galactosylceramide, which represents an NKT-specific ligand. They also reported that CD1d-deficient dnTGFβRII mice had decreased hepatic lymphoid cell infiltrates and milder cholangitis compared with controls.90

We are well aware that innate immune system hyper-responsiveness is likely not sufficient for immune tolerance breakdown. We can hypothesize, however, that these alterations might play a role in the initiation and/or perpetuation of autoimmune injury. This is particularly intriguing considering the previously mentioned study by Mattner et al., who demonstrated that in a murine model of PBC, N. aromaticivorans was capable of inducing autoreactive AMAs and chronic T-cell-mediated autoimmunity against small bile ducts in an NKT-dependent fashion.43

Animal models

Similar to all complex diseases, such as autoimmune disease, the development of an animal model is of obvious importance in elucidating the mechanism(s) responsible for the initiation and progression of disease.

Several models, mostly murine,91 have been proposed for PBC and are illustrated in Table 4. Several mouse strains have been reported to be spontaneous PBC model animals and will be briefly described.

Table 4 Similarities of the innate and acquired immunity compartments between proposed animal models and human PBC

Two animal models, i.e., dnTGFβRII and an IL-2 receptor-α knockout mouse, indicate the possible crucial role of Treg deficiency in loss of immune tolerance with the consequent development of an autoimmune response against PDC-E2 in PBC. A mouse with dnTGFβRII showed PBC-like liver disease, i.e., 100% AMA positivity against PDC-E2.92 Transforming grown factor-β receptor II is essential for signal transduction of transforming grown factor-β, which is a key regulator of lymphocyte activation.93

A mouse deficient for IL-2 receptor-α, which is highly expressed on Tregs, developed 100% AMA positivity against PDC-E2, 80% ANA positivity, and lymphocyte infiltration around the portal tracts associated with cholangiocyte injury.94

Animal models support our hypothesis that xenobiotics can induce loss of tolerance. First, our group demonstrated loss of tolerance in rabbits immunized with 6-bromohexonate, a xenobiotically modified hapten mimicking lipoic acid, coupled with bovine serum albumin. The immunized rabbits were able to produce not only antibodies against the xenobiotic, but also a high titer of anti-PDC-E2 antibodies. Anti-PDC-E2 antibodies in this model were not, however, sufficient to induce specific hepatic lesions, at least in the short term.95 More recently, induction of PBC-like lesions was obtained in a non-obese diabetic (NOD) background by Wakabayashi et al. and in guinea-pigs exposed to xenobiotic immunization by Leung et al.96, 97

An additional animal model is a variant of the NOD mouse model (NOD.c3c4). It has been described that NOD.c3c4 presents autoimmune cholestasis and PBC-specific serology, showing AMA positivity of 50–60% and ANA positivity of 80–90%. Histologically, NOD.c3c4 presents lymphocyte infiltration around portal tracts with chronic non-suppurative destructive cholangitis and epithelioid granuloma formations; nevertheless, the morphological features of bile ducts differ somewhat from those in human PBC.98

PBC pathogenesis

Once we have illustrated the available evidence on the immune mechanisms involved in PBC pathogenesis, it should be clear that the etiology of the tissue injury and, in particular, the outstanding organ specificity of the immune-mediated tissue damage, remains to be elucidated, and several hypotheses have been proposed. We are convinced that these should not be regarded as mutually exclusive from any causative factor, such as susceptible genes, but rather as terminal mechanisms of the pathway leading to the clinical manifestations, possibly mediated by a specific cytokine pattern.99

One major hypothesis for the selective destruction of biliary epithelial cells implies that the immunodominant autoantigen PDC-E2 should be aberrantly exposed on the cholangiocyte cell surface, where it may be recognized by AMA and/or antigen-specific T cells. Studies based on in situ hybridization of PDC-E2 mRNA failed to demonstrate significant differences in its amount in PBC liver compared with other liver diseases. PDC-E2 may be selectively overexpressed in small bile duct cholangiocytes, as indirectly suggested by early experimental evidence showing positive staining of a murine anti-PDC-E2 monoclonal antibody selectively on the surface of biliary epithelial cells in the livers of patients with PBC, but not in normal controls.

On the other hand, co- or post-translational modifications of PDC-E2 may cause its abnormal turnover, leading to its accumulation. Chemicals (i.e., xenobiotics) disposed of by the liver may have a role in this scenario by accumulating in the biliary epithelial cells and modifying PDC-E2 locally. Solid data to support these fascinating mechanisms are lacking or weak and we cannot rule out that the molecules expressed and identified on the ductular surface and recognized by AMA may not be PDC-E2 itself, but possibly unrelated PDC-E2 mimics crossreacting with human PDC-E2.

As mentioned before, the pathogenetic role of AMA is not yet clear. AMA belongs mainly to the immunoglobulin G isotype, in particular immunoglobulin G3 subclasses, and thus is potentially pathogenic through different mechanisms, e.g., complement activation and antibody-dependent cytotoxicity. There is no direct experimental evidence, however, supporting the involvement of these mechanisms in the pathogenesis of PBC.

AMA can be also of the immunoglobulin A (IgA) isotype. The role of such AMA-IgA has long been ignored, but may be critical in the pathogenesis of PBC. AMA-IgA can indeed be detected not only in sera, but also in bile, saliva and urine of patients with PBC, in some cases correlating with disease severity. Moreover, IgA represents the principal immunoglobulin isotype on epithelial surfaces, including biliary epithelia. AMA-IgA has been reported to colocalize with PDC-E2 both inside the cell cytoplasm and in the apical membrane of cholangiocytes in PBC, but not in controls. Thus, AMA-IgA and in particular AMA-IgA bound to a mitochondrial antigen could disrupt cell metabolism and may also induce cellular dysfunction and damage, thus leading to a tissue-specific injury. We cannot preclude the possibility that the apical staining obtained with anti-PDC-E2 monoclonal antibodies may be secondary to the presence of immune complexes formed by secreted IgA and AMA antigens, as one line of evidence seems to suggest.100

Apoptosis of biliary epithelial cells in PBC warrants further discussion and may prove crucial for immune tolerance breakdown,63, 101 as illustrated under other conditions. Quite surprisingly, Odin et al. first demonstrated that PDC-E2 remains intact and retains its immunogenicity during cholangiocyte apoptosis because of a cell-specific lack of glutathionylation of biliary epithelial cells.61 The intact PDC-E2 in apoptotic fragments could then be taken up by local antigen-presenting cells and transferred to regional lymph nodes for priming of cognate T cells, thus initiating PBC. This is indeed an attractive possibility; however, solid data of such antigen presentation are lacking, and we cannot exclude the possibility that the reported mechanisms are not PBC-specific.

In conclusion, we may now recapitulate our current working hypothesis (Figure 1). Three major events in PBC are represented, i.e., bile duct cell apoptosis, female predominance and genetic susceptibility. A mimicking microorganism (possibly the ubiquitous N. aromaticivorans) enters the human system through the digestive mucosa, and its PDC-E2-like proteins are modified within the liver by xenobiotics to form immunoreactive adducts. These modifications could then be sufficient to trigger the innate immune system to initiate a cascade of local inflammatory events, resulting in local dendritic cell activation and antigen processing. Mucosal antigen-presenting cells could in turn activate autoreactive T and B cells that are directed to the liver through the portal system. T cells, therefore, could participate directly not only in autoimmune injury, but also in its amplification and perpetuation. B cells, on the other hand, could secrete AMA, particularly of the IgA type. AMA-IgA could then be transported to the vascular side of biliary epithelial cells, where they could recognize PDC-E2-like molecules located on the lumenal surface cell membrane. AMA-IgA/PDC-E2-like molecule engagement could initiate an apoptotic signaling cascade. Ultimately, the immune complexes of postapoptotic PDC-E2 and immunoglobulin G-AMA and the direct cytopathic effects of autoreactive T cells (and possibly AMA) lead to selective bile duct destruction.

Figure 1
figure1

A mechanistic view of the etiopathogenetic hypothesis of PBC development. In PBC, the autoimmune response against biliary epithelial cells is secondary to the triggered immune tolerance breakdown to PDC-E2. (a) Different factors have been implicated for immune tolerance breakdown. Infectious agent(s) sharing sequence homology with PDC-E2, i.e., N. aromaticivorans, can induce immune crossreactivity with PDC-E2. (b) Apoptosis of cholangiocytes can lead to the accumulation of structurally intact PDC-E2, which therefore retains its immunogenicity. (c) Xenobiotics (2-nonynoic acid) disposed of by the liver can accumulate within cholangiocytes and potentially modify PDC-E2 locally. (d) Defective Tregs may lead to an inappropriate control of T-cell response against PDC-E2. (e) Major sex chromosome defects, e.g., X monosomy, have been implicated to explain female predominance of immune tolerance breakdown in autoimmunity in general and in PBC in particular. (f) Genetic factors participate in conferring individual susceptibility, predisposing immune tolerance breakdown, while epigenetic factors can explain phenotype/genotype discordance. Once breakdown of tolerance has been established, innate immunity and adaptive immunity, both humoral and cellular, are thought to be involved in liver tissue damage. PBC, primary biliary cirrhosis; PDC-E2, pyruvate dehydrogenase complex E2.

Future developments

What does the future hold for PBC etiology and pathogenesis? We are convinced that efforts should be dedicated to overcoming some of the conceptual and logistic difficulties.

First, we encourage the collection of representative families through a worldwide effort to further confirm reported associations. This will empower definition of the genetic basis associated with PBC through the collection of a large series of patients and controls and the use of genome-wide analysis on thousands of single nucleotide polymorphisms. Second, the role of xenobiotics and bacteria in the onset of PBC should be further studied by means of new molecular multiplex tools. Third and most important, it is time to prove the pathogenic role of AMA in PBC. In this view, the proposed animal models may be a good starting point to achieve this goal. Finally, we are convinced that future progress in PBC will be possible only through a multidisciplinary approach uniting clinicians, basic immunologists, geneticists, chemists and microbiologists.

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Correspondence to Carlo Selmi.

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Selmi, C., Meda, F., Kasangian, A. et al. Experimental evidence on the immunopathogenesis of primary biliary cirrhosis. Cell Mol Immunol 7, 1–10 (2010). https://doi.org/10.1038/cmi.2009.104

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