Aetiopathogenesis of autoimmune hepatitis

Journal of Autoimmunity 34 (2010) 7–14

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Journal of Autoimmunity journal homepage: www.elsevier.com/locate/jautimm

Review

Aetiopathogenesis of autoimmune hepatitis Maria Serena Longhi a, Yun Ma a, Giorgina Mieli-Vergani a, b, Diego Vergani a, * a b

Institute of Liver Studies, King’s College London School of Medicine at King’s College Hospital, Denmark Hill, London SE5 9RS, UK Paediatric Liver Centre, King’s College London School of Medicine at King’s College Hospital, Denmark Hill, London SE5 9RS, UK

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 August 2009 Accepted 24 August 2009

Interface hepatitis, histological hallmark of autoimmune hepatitis (AIH), is a dense portal mononuclear cell infiltrate invading the parenchyma, consisting of CD4 and CD8 T-lymphocytes, monocytes/macrophages and plasma cells. What triggers AIH is unknown, but there is evidence that failure of immune homeostasis plays a permissive role allowing liver autoantigen-specific cells to attack hepatocytes. Damage is likely to be orchestrated by CD4 T-lymphocytes recognizing an autoantigenic liver peptide. For autoimmunity to arise, the peptide, embraced by an HLA class II molecule, must be presented to naı¨ve CD4 T-helper (TH0) cells by professional antigen-presenting cells. Once activated, TH0 cells can differentiate into TH1 cells, which are pivotal to macrophage activation, enhance HLA class I expression, rendering liver cells vulnerable to CD8 T-cell attack, and induce HLA class II expression on hepatocytes; or into TH2 cells producing IL-4, IL-10 and IL-13, cytokines favouring autoantibody production by B-lymphocytes. Autoantigen recognition is tightly controlled by regulatory mechanisms, such as those exerted by CD4þCD25þ regulatory T-cells (T-regs). Numerical and functional T-reg impairment characterizes AIH, particularly during active disease. Advances in the study of autoreactive T-cells stem mostly from AIH type 2, where the main autoantigen, CYP2D6, is known enabling characterization of antigen-specific immune responses. Monocyte involvement in the autoimmune liver attack has been recently reported. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Liver Autoantibody Humoral immunity Cellular immunity Immune regulation

1. Introduction

2. Genetics

Autoimmune hepatitis (AIH) is an inflammatory liver disease, characterized by female preponderance, elevated serum transaminase activity, positive organ and non-organ-specific autoantibodies, elevated immunoglobulin G (IgG) and a histological picture of interface hepatitis. According to the nature of autoantibodies detected at the time of diagnosis, two types of AIH are recognized: type 1 is characterized by anti-nuclear (ANA) and/or anti-smooth muscle (SMA) antibodies; type 2 by anti-liver kidney microsomal type 1 (anti-LKM-1) antibody. The aetiology of AIH is unknown, though both genetic and environmental factors are involved in its expression. Immune reactions against host liver antigens are believed to be the major mechanism of liver damage.

AIH is a ‘complex trait’ disease, i.e. a condition not inherited in a Mendelian autosomal dominant, autosomal recessive or sexlinked fashion. The mode of inheritance of a complex trait disorder is unknown and involves one or more genes, operating alone or in concert, to increase or reduce the risk of the trait, and interacting with environmental factors. Susceptibility to AIH is conferred by genes within the human leukocyte antigen (HLA) region on the short arm of chromosome 6, especially those encoding DRB1 alleles. Since the role of class II major histocompatibility complex (MHC) molecules (HLA in man) is to present peptide antigens to CD4 T-cells, this suggests the involvement of HLA class II antigen presentation and T-cell activation in the pathogenesis of AIH. In Europe and North America, susceptibility to AIH type 1 is conferred by the possession of HLA-DR3 (DRB1*0301) and DR4 (DRB1*0401), both heterodimers containing a lysine residue at position 71 of the DRB1 polypeptide and the hexameric amino acid sequence LLEQKR at positions 67–72 [1,2]. In Japan, Argentina and Mexico, susceptibility is linked to DRB1*0405 and DRB1*0404, alleles encoding arginine rather than lysine at position 71, but sharing the motif LLEQ-R with DRB1*0401 and DRB1*0301 [3]. Thus,

Abbreviations: AIH, autoimmune hepatitis; ANA, anti-nuclear antibodies; SMA, anti-smooth muscle antibodies; anti-LKM-1, anti-liver kidney microsomal type 1 antibody; APECED, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy; T-regs, CD4þCD25þ regulatory T-cells; CYP2D6, cytochrome P450IID6; CTL, cytotoxic T-cells. * Corresponding author. Tel.: þ44 203 2993305; fax: þ44 203 2993700. E-mail address: [email protected] (D. Vergani). 0896-8411/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaut.2009.08.010

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K or R at position 71 in the context of LLEQ-R may be critical for susceptibility to AIH, favouring the binding of autoantigenic peptides, complementary to this hexameric sequence. However, an alternative model based on valine/glycine dimorphism at position 86 of the DR-beta polypeptide has been proposed, better representing the key HLA associations in patients from Argentina and Brazil [1,2]. In a study from Japan, patients with AIH type 1 were found to have DRB1 alleles which encode histidine at position 13 [1,2]. There appears therefore to be at least three models, suggesting that different genetic associations are present in different populations and that the peptides presented by HLA class II molecules to the T-cell receptors are different and may derive from different antigens. Thus, these HLA associations may be the molecular footprints of the prevailing environmental triggers that precipitate AIH type 1 in different environments. In this context, it is of interest that in South America possession of the HLADRB1*1301 allele, which predisposes to paediatric AIH type 1 in that population, is also associated with persistent infection with the endemic hepatitis A virus. The lysine-71 and other models for AIH type 1 cannot explain completely the disease, since for example in European and North American patients presence of lysine-71 is associated with a severe, mainly juvenile, disease in those DRB1*0301 positive, but to a mild, late onset, disease in those DRB1*0401 positive. Susceptibility to AIH type 2 is conferred by the possession of HLA-DR7 (DRB1*0701) and DR3 (DRB1*0301), patients positive for DRB1*0701 having a more aggressive disease and severe prognosis [4]. A meta-analysis study, recently conducted by Duarte-Rey et al. to evaluate the contribution of HLA class II alleles to AIH in Latin America, has reported that HLA DQ2 and HLA DR52 are risk factors, while HLA DR5 and HLA DQ3 are protective factors in this patient population [5]. Further analysis performed at the allelic level has indicated that the possession of DQB1*02, DQB1*0603, DRB1*0405 and DRB1*1301 predisposes to, while that of DRB1*1302 and DQB1*0301 protects against AIH occurrence. The study has then confirmed that the susceptibility to AIH, conferred by HLADRB1*1301 and DRB1*0301, is possibly associated with the presence of valine at the position 86 of the DR-beta polypeptide. In contrast the protective allele DRB1*1302 was found to differ from the DRB1*1301 for the presence of glycine instead of valine in this position [5]. Genetic studies looking outside MHC genes have identified the cytotoxic T-lymphocyte associated antigen 4 (CTLA-4), an adhesion molecule that down-regulates peripheral T-cell immune responses, as a possible non-MHC gene predisposing to autoimmune disease. CTLA-4 gene polymorphisms involving a transition from adenine (A) to guanine (G) in exon 1 at position 49 confer susceptibility to a number of autoimmune conditions, including AIH type 1 where the genotype distribution is significantly different in patients in comparison to normal controls, with 32% of patients bearing the CTLA-4*A,A genotype, 54% the CTLA-4*A,G genotype and 14% the CTLA-4*G,G genotype, compared to 50%, 30% and 13% in the normal controls [6]. This finding, however, was not confirmed in Brazilian

patients with AIH type 1 and 2 [7]. Polymorphism of the TNF-a gene may also confer susceptibility to AIH, Czaja et al. having reported that polymorphism at position 308 in its promoter region is associated with severity of AIH type 1 in Europe and North America in synergy with DRB1*03 [8], a finding, however not present in Japanese patients [9]. A form of AIH resembling AIH type 2 affects some 20% of patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a condition also known as autoimmune polyendocrine syndrome 1. APECED is a monogenic autosomal recessive disorder caused by homozygous mutations in the AIRE1 gene and characterized by a variety of organ-specific autoimmune diseases, the most common of which are hypoparathyroidism and primary adrenocortical failure, accompanied by chronic mucocutaneous candidiasis [10,11]. The AIRE1 gene sequence consists of 14 exons containing 45 different mutations, with a 13 bp deletion at nucleotide 964 in exon 8 accounting for more than 70% of APECED alleles in the UK [10]. The protein encoded by AIRE1 is a transcription factor. AIRE1 is highly expressed in medullary epithelial cells and other stromal cells in the thymus involved in clonal deletion of self-reactive T-cells [12]. Studies in a murine model indicate that the gene inhibits organ-specific autoimmunity by inducing thymic expression of peripheral antigens in the medulla leading to central deletion of autoreactive T-cells. Interestingly, APECED has a high level of variability in symptoms, especially between populations. Since various gene mutations have the same effect on thymic transcription of ectopic genes in animal models, it is likely that the clinical variability across human populations relates to environmental or genetic modifiers. Of the various genetic modifiers, perhaps the most likely to synergize with AIRE mutations are polymorphisms in the HLA region. HLA molecules are not only highly variable and strongly associated with multiple autoimmune diseases, but are also able to affect thymic repertoire selection of autoreactive T-cell clones. Carriers of a single AIRE mutation do not develop APECED. However, although the inheritance pattern of APECED indicates a strictly recessive disorder, there are anecdotal data of mutations in a single copy of AIRE being associated with human autoimmunity of a less severe form than classically defined APECED [10,11]. The role of AIRE1 heterozygote state in the development of AIH remains to be established. AIRE1 mutations have been reported in 3 children with severe AIH type 2 and extrahepatic autoimmune manifestations [13] and in 4 children with AIH type 1 and a family history of autoimmune disease [14]. 3. Immunopathogenesis 3.1. Impairment of T-regulatory cells The mechanisms underlying the breakdown of self-tolerance in autoimmune disease have not been fully elucidated, though there is mounting evidence that a defect in homeostatic processes, normally keeping the response to self-antigens under control, is involved (Table 1). Early studies have shown that patients with AIH

Table 1 Evidence of impaired immune regulation in AIH. AIH-1      

Impaired suppressor cell function corrected by in vitro exposure to steroids [15] Defect in a subset of T-cells controlling responses to a liver-specific membrane autoantigen [16] Numerical and functional impairment of CD4þCD25þ T-regs [17,18] T-regs defective at regulating CD4 and CD8 T-cell proliferation and IFN-g production [17,18] T-regs defective at promoting secretion of regulatory cytokines by their targets [19] T-regs unable to restrain monocyte activation and function [23]

T-regs: regulatory T-cells; IFN-g: interferon-gamma.

AIH-2  Numerical and functional impairment of CD4þCD25þ T-regs [17,18]  T-regs are defective in regulating CD4 and CD8 T-cell proliferation and IFN-g production [17,18]

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have impaired ‘suppressor’ cell function, which could be corrected following in vitro exposure to therapeutical doses of steroids [15]. Later on a defect specifically involving a subpopulation of T-lymphocytes controlling immune responses directed against a liver-specific membrane autoantigen was reported [16]. In line with these observations, evidence provided over the last five years has indicated that an impairment in regulatory T-cells is key to the loss of immune tolerance in AIH and to the emergence of uncontrolled effector autoimmune responses [17–19]. Among recently described regulatory T-cell subpopulations, namely natural killer T (NKT) cells, T-helper 3 (Th3), T-regulatory 1 (Tr1), CD8þCD28 and gd T-cells, the CD4 T-lymphocytes constitutively expressing the IL-2 receptor (IL-2R) a chain (CD25) have emerged as a major subset, being central to immune tolerance maintenance [20]. CD4þCD25þ regulatory T-cells (T-regs), which in health constitute 5–10% of the total population of peripheral CD4 T-lymphocytes control innate and adaptive immune responses by preventing proliferation and effector function of autoreactive T-cells. Studies mainly conducted in the in vitro setting have indicated that T-regs can suppress their targets directly or through antigen-presenting cells [21]. In addition to CD25, also present on activated T-cells, T-regs express a number of defining markers, including the glucocorticoid inducible tumour necrosis factor receptor (GITR), CTLA-4 and the forkhead winged helix transcription factor box P3 (FOXP3), the best marker for murine T-regs and still the most specific human T-reg marker, being associated with the ability of these cells to suppress [22]. In AIH, T-regs are defective in number, this defect being more marked at disease presentation than during drug-induced remission, when a partial T-reg restoration is observed [17,18]. In a study involving forty-one children with AIH, 30 ANAþ/SMAþ and 11 LKM-1þ, T-reg frequency inversely correlated with markers of disease severity, such as levels of antibody to soluble liver antigen (SLA) (see below) and anti-LKM-1 autoantibody titres, suggesting that T-reg reduction favours the serological manifestations of autoimmune liver disease [17]. Functional studies have demonstrated that, at variance with healthy subjects, T-regs isolated from children with AIH are unable to regulate proliferation and IFN-g production by CD4 and CD8 T-cells, both involved in the autoimmune liver attack [17,18]. Akin to cell number, T-reg suppressor function improves during druginduced remission, though never reaches levels seen in health. Autologous cross-culture experiments suggest that loss of control over T-cell effector function in AIH is due to a primary T-reg defect and not to target unresponsiveness [18]. T-regs from AIH patients are defective at promoting regulatory cytokine secretion by their targets, which in health create a regulatory environment supporting and enhancing the function of T-regs themselves [19]. In addition to impaired regulation of effector T-cell function, T-regs in AIH are unable to restrain, and even enhance, the activation of monocytes, cells of the innate immune system abundantly present in the portal-periportal inflammatory infiltrate [23]. Because of their ability to control autoaggression, T-regs represent an attractive candidate for immune intervention aiming at reconstituting self-tolerance in autoimmune conditions [24]. The immunotherapeutic use of CD4þCD25þ T-regs, however, has been so far hindered by their limited ability to proliferate and by their propensity to apoptosis [25], making difficult their expansion to numbers adequate for treatment. The observation of a partial T-reg restoration observed in patients during remission [17,18] indicates that T-regs in AIH, though impaired, have the potential to expand and regain their function. Following in vitro exposure to a polyclonal T-cell stimulation, T-regs can be expanded not only in healthy subjects, but also in patients with AIH [26]. While

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maintaining the phenotypic features of original CD4þCD25þ T-cells, expanded T-regs increase their FOXP3 expression alongside their suppressor function. Using the same approach, in AIH T-regs were also generated de novo and expanded from the pool of CD4þCD25 T-cells [26], a heterogeneous population mainly composed of effector cells but also including a subset of lymphocytes with regulatory potential. Whether T-regs with autoantigen specificity, shown in animal studies to suppress more efficiently than their non-antigen-specific counterpart, would represent an immunotherapeutic option also in AIH patients, is still under investigation. In this regard, AIH type 2 would be an excellent model for attempting reconstitution of selftolerance by specific immunological intervention since not only is the key autoantigen, i.e. cytochrome P450IID6 (CYP2D6), known [27] but also the antigenic regions (CYP2D6217–260 and CYP2D6305– 348), target of B, CD4 and CD8 T-cell immune responses well characterized [4,28,29]. Generation of T-regs specifically controlling effector immune responses to CYP2D6 would be critical for reestablishing immune tolerance in AIH type 2 and therefore achieving earlier disease remission, while limiting or maybe even preventing life-long immunosuppression. 3.2. Molecular mimicry Another mechanism that has been implicated in the initiation of autoimmune damage is ‘‘molecular mimicry’’ whereby immune responses to external pathogens become directed towards structurally similar self-components. Several studies have shown that autoimmunity is a common feature during chronic HBV and HCV infections, where up to 50% of the patients become seropositive for autoantibodies such as ANA and SMA [30,31]. It is also well documented that positivity for LKM-1 is present in up to 10% of patients chronically infected with HCV and correlates with disease severity and adverse reactions to interferon treatment [32,33]. These findings have led to postulate a role for HBV and HCV infection in the initiation of the autoimmune process, though a strong link between autoimmunity and viral hepatitis has been shown only for HCV. LKM-1 autoantibodies recognise linear epitopes within cytochrome CYP2D6, a member of the hepatic P450 cytochrome family and, as mentioned above, the main target antigen in AIH type 2. Interestingly, reactivity against CYP2D6193–212, identified as major B-cell epitope in AIH type 2, being recognized by 93% of the patients, is also found in 50% of HCV infected patients with seropositivity for LKM-1 autoantibodies [34]. HCVþ/LKM-1þ patients have antibodies that cross-react with homologous regions of HCV (NS5B HCV2985–2990) and CYP2D6 (CYP2D6204–209), and also of cytomegalovirus (exon CMV130–135) [28]. Cross-reactive mechanisms to explain the emergence of CYP2D6-specific autoimmunity have also been suggested for other sequences of CYP2D6 which share homologies with HCV and Herpes simplex virus (HSV), such as the sequence spanning aa 310–324 of E1 HCV and aa 156–170 of IE175 HSV1, which share homology with the CYP2D6 region comprising aa 254–271. As anti-LKM-1 antibodies cross-react with homologous regions of CYP2D6, HCV, HSV, and CMV, a ‘multi-hit’ mechanism for the generation of these antibodies and possibly of AIH type 2 may be envisaged. In this model, multiple exposures to CMV or HSV, common viral pathogens, may establish permissive immunological conditions, by priming a cross-reactive subset of T-cells, in a genetically predisposed host. Depending on the degree of immunological priming, i.e. level of exposure, and the degree of genetic susceptibility (particularly at the HLA locus and coding regions for ‘innate’ components of immunity), a minority of recurrently infected individuals may progress to autoimmune disease. It is therefore conceivable that an as yet unsuspected virus infection may be part of the origin of the autoimmune attack in AIH.

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Evidence for such a mechanism has been provided by an interesting case-report describing a 10-year-old girl who acquired HCV infection after liver transplantation for end-stage liver disease caused by a1-antitrypsin deficiency [35]. Two weeks after HCV infection IgM anti-LKM-1 appeared, followed by IgG anti-LKM-1, a finding suggesting HCV as a trigger of a primary anti-LKM-1/anti-CYP2D6 autoimmune response. That HCV is involved in immune tolerance breakdown has been also postulated by a recent study by Agmon-Levin et al., who reported that serum anti-HCV antibodies were found in up to 8.7% of patients with autoimmune diseases, including cryoglobulinaemia, mixed cryoglobulinaemia, Hashimoto’s thyroiditis and inflammatory bowel disease [36]. 3.3. Mechanisms leading to autoimmune liver damage Regardless of the factors triggering the autoimmune process, the pathogenic mechanism leading to the liver damage operates in a complex scenario, which involves the intervention of both innate and adaptive immune responses. The histological picture of interface hepatitis (Fig. 1), with its striking infiltrate of lymphocytes, plasma cells, and monocytes/macrophages was the first to suggest an autoaggressive cellular immune attack in the pathogenesis of AIH. Immunohistochemical studies, focused on the phenotype of the inflammatory cells infiltrating the liver parenchyma in AIH, have revealed a predominance of ab T-cells [37]. Amongst these cells, a majority was found to be positive for the CD4 helper/inducer phenotype and a sizeable minority for the CD8 cytotoxic/ suppressor phenotype. Lymphocytes of non-T-cell lineage included NK cells, monocytes/macrophages and B-lymphocytes [37]. Liver damage is believed to initiate with the presentation of a self-antigenic peptide to the T-cell receptor (TCR) of an uncommitted T-helper (TH0) lymphocyte by professional antigen-presenting cells (APCs), such as macrophages, dendritic cells and B-lymphocytes, in the presence of co-stimulating signals, induced by the interaction of CD28 on TH0 and CD80 on APC. Once exposed to the antigen embraced by an HLA class II molecule on the APC, the TH0 lymphocytes become activated and, according to the presence in the milieu of IL-12 or IL-4, they differentiate into TH1 or TH2 cells and initiate a series of immune reactions determined by the cytokines they produce. TH1 cells predominantly secrete IL-2 and IFN-g,

Fig. 1. The portal and periportal inflammatory infiltrate characteristic of autoimmune hepatitis is composed of lymphocytes, monocytes/macrophages and plasma cells (interface hepatitis, arrows). Haematoxylin & eosin staining. (Picture kindly provided by Dr Alberto Quaglia.)

the latter being the main orchestrator of tissue damage because of its ability to stimulate cytotoxic T-cells (CTL), to enhance the expression of HLA class I molecules on APCs and of HLA class II molecules on hepatocytes [38,39] and activate monocytes/macrophages, which in turn release IL-1 and tumour necrosis factor alpha (TNF-a). The expression of HLA class II on hepatocytes, that normally do not express it, enables them to present the autoantigen to TH1 lymphocytes and hence the perpetuation of the autoimmune process. The function of TH1 cells is counterbalanced by that of TH2 cells, arising in the presence of IL-4 and mainly producing IL-4, IL10 and IL-13. These cytokines induce also the maturation of B-cells into plasma cells, with consequent production of autoantibodies. If regulatory cells are numerically deficient and/or impaired in their suppressor function, these effector responses are perpetuated with ensuing persistent liver cell destruction by the direct action of CTL, cytokines released by TH1 cells and monocytes/macrophages, complement activation or engagement of natural killer cells by the autoantibodies bound to the hepatocyte surface. The role of the recently described TH17 cells, a cell subset that arises in the presence of transforming growth factor beta (TGF-b) and IL-6 and shares the same CD4 progenitors as T-regs [40], is under investigation. Mechanisms leading to and perpetuating autoimmune attack in AIH are depicted in Fig. 2. The contribution of both humoral and cellular immune responses to AIH pathogenesis is reviewed below. 3.4. Humoral immunity Table 2 summarizes findings related to the role of humoral immunity in the pathogenesis of AIH. The role of autoantibodies in the pathogenesis of autoimmune liver damage has been suggested by the finding that hepatocytes, isolated from patients with AIH, are coated with immunoglobulins and are susceptible to cytotoxicity when exposed to autologous Fc receptor-bearing mononuclear cells [41,42]. It has been also shown that titres of anti-liver-specific membrane lipoprotein (LSP) antibody, as well as antibodies to the LSP components asialoglycoprotein receptor (ASGPR) and alcohol dehydrogenase (ADH), correlate with biochemical and histological indices of disease severity [43–45]. Anti-SLA antibodies, targeting the UGA suppressor tRNAassociated antigenic protein (tRNP(Ser)Sec) recently re-named as SepSecS [46] and originally described by Manns [47], are found in some 50% of patients with type 1 and type 2 AIH, where they define a disease course more severe than in seronegative patients [48], characterized by more aggressive histology, longer time to remission, high tendency to relapse and to progress to end-stage liver disease requiring transplantation. The finding that CYP2D6 is expressed on the surface of hepatocytes, and therefore susceptible to be recognized by LKM-1 autoantibodies, has led to the hypothesis that these autoantibodies could be directly involved in the pathogenesis of autoimmune liver damage in AIH type 2 [49]. Following the identification of CYP2D6 as the target antigen of LKM-1, the search for B-cell epitopes on CYPD26 has been the focus of intensive investigation. In AIH type 2 anti-LKM-1 antibodies recognise linear regions (autoepitopes) of CYP2D6 in a hierarchical manner. Thus, the principal linear B-cell epitope, CYP2D6193–212 is recognized by 93% of patients, CYP2D6257–269 by 85%, CYP2D6321–351 by 53%, and two additional minor epitopes CYP2D6373–389 and CYP2D6410–429 are recognized by 7% and 13% respectively [28]. Using two complementary strategies, consisting in the expression of both the full-length and a series of truncated CYP2D6 proteins in a eukaryotic system, and in the characterization of the identified epitope by mutagenesis, Ma and colleagues have provided a conformational (i.e. highly dependent on the three-dimensional structure of the protein) epitope mapping of CYP2D6 [50], showing

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NK NK APC APC Peptide

Liver Liver cell cell

Class II

Co - stimuli

Class II

Th0 Th0

Th1 Th1

IL-4

M M P P

IFN-γ IL-4 IL-10

IL-17

TNF- α

CTL CTL

IL-2

Th2 Th2

TGF-β

IL-1

Class I

IFN- γ

IL-12

Tr Tr

IL-17

IL-13 IL-6

B B

Th17 Th17 Ts

Fig. 2. Autoimmune attack to the liver cell. A specific autoantigenic peptide is presented to an uncommitted T-helper (TH0) lymphocyte within the HLA class II molecule of an antigen-presenting cell (APC). TH0 cells become activated and, according to the presence in the microenvironment of interleukin (IL)-12 or IL-4 and the nature of the antigen, differentiate into TH1 or TH2 and initiate a series of immune reactions determined by the cytokines they produce: TH1 secrete IL-2 and interferon-gamma (IFN-g), which stimulate cytotoxic T-lymphocytes (CTL), enhance expression of class I and induce expression of class II HLA molecules on hepatocytes and activate macrophages; activated macrophages release IL-1 and tumour necrosis factor alpha (TNF-a). TH2 secrete mainly IL-4, IL-10 and IL-13, and direct autoantibody production by B-lymphocytes. If regulatory T-cells (Tr) do not oppose, a variety of effector mechanisms are triggered: liver cell destruction could derive from the action of CTL; cytokines released by TH1 and recruited macrophages; complement activation or engagement of Fc receptor-bearing cells such as natural killer (NK) lymphocytes by the autoantibody bound to the hepatocyte surface. The role of the recently described TH17 cells, which arise in the presence of transforming growth factor beta (TGF-b) and IL-6, is under investigation.

that the antigenicity was confined to the C terminal portion of the molecule where it increased stepwise towards the C terminal. In addition, a short amino acid sequence, CYP2D6316–327, further characterized by molecular modelling, was noted to be located on the surface of the molecule, suggesting that it may be directly involved in autoantibody mediated liver cell damage [50]. 3.5. Cellular immunity Evidence implicating cellular immunity in the pathogenesis of AIH is summarized in Table 3. Early investigations of cellular immune mechanisms involved in the pathogenesis of autoimmune liver damage led to the conclusion that patients suffering from AIH had circulating lymphocytes ‘‘sensitised’’ to liver antigens and able to kill hepatocytes in vitro. Studies performed using separated T- and non-T-cell subsets from the peripheral blood of AIH patients and autologous hepatocytes as targets demonstrated that cytotoxic cells were present in the nonT-cell subpopulation [41]. The authors also observed high cytotoxic activity in all untreated patients with AIH but in only 40% of those in remission induced by immunosuppressive treatment [41]. The findings that the cytotoxic cells were present in the non-T-cell compartment and that aggregated IgG were able to block the cytotoxic activity led to the suggestion that an antibody-dependent-cell-mediated cytotoxicity (ADCC) is involved in the pathogenesis of the autoimmune liver damage, a hypothesis confirmed

later by the observation that hepatocytes from AIH patients carrying IgG on their surface were susceptible to damage by lymphocytes from healthy subjects [42]. Subsequent studies of clonal analysis, performed in the early 1990s, showed that cytotoxicity against liver-specific antigens could also be detected within the T-cell compartment [51]. Studies conducted later to identify liver-specific T-cells in peripheral blood of children with AIH demonstrated that patients have a 10-fold higher frequency of liver antigen-specific precursors in their circulation than normal subjects [52]. Experiments of clonal analysis, performed by the same authors to investigate the function of activated T-lymphocytes in AIH, revealed a high frequency of CD4 T-cell clones expressing the HLA-DR molecule. Inhibition of proliferation with anti-HLA-DR and anti-CD4 monoclonal antibodies led to the conclusion that these clones follow the classical rules of immune recognition, being able to recognise antigens in the context of class II HLA molecules [51]. Subsequent investigations showed that in AIH the largest number of clones generated from the peripheral blood are CD4þ T-cells bearing the ab T-cell receptor (TCRab), while the highest proportion of clones obtained from liver biopsies, are CD4CD8 T-cells bearing the gd T-cell receptor (TCRgd) or CD8þ ab T-cells. Both types of liver derived clones were able to proliferate in response to liver-specific antigens, such as ASGPR and ADH; clones that proliferated in the presence of ASGPR, were also able to induce the production of ASGPR-specific autoantibodies from B-lymphocytes in vitro [52].

Table 2 Humoral immune mechanisms involved in the pathogenesis of AIH. AIH-1

AIH-2

 Anti-SLA, targeting UGA serine transfer RNA (tRNA)-associated protein [61], is present in 60% of patients with type 1 AIH and defines a particularly severe disease course [48]. SLA: soluble liver antigen; CYP2D6: cytochrome P4502D6.

 Anti-LKM-1 characterizes type 2 AIH and targets CYP2D6.  Anti-LKM-1 recognises 5 linear CYP2D6 epitopes (CYP2D6193–212, -257–269, -321–351, -373–389 and -410–429) [28] and  1 conformational CYP2D6 epitope (CYP2D6316–327) [50]  Anti-SLA is present in 40% of patients with type 2 AIH and defines a particularly severe disease course [48].

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Table 3 Cellular immune effector mechanisms involved in the pathogenesis of AIH. AIH-1

AIH-2

Adaptive immunity

 Non-T-cell cytotoxicity associated with hepatocyte damage [41]  T-cell cytotoxicity directed to liver-specific antigens [51]  Most clones from peripheral blood are CD4 T-cells TCRabþ; while clones from liver are CD4CD8 TCRgdþ or CD8 TCRabþ and both react to ASGPR and ADH [52]  Cytotoxicity to SLA to be tested

Innate immunity (monocytes)

 Monocytes exhibit a vigorous spontaneous migration, are skewed towards a pro-inflammatory phenotype (high TNF-a/IL-10) and express elevated levels of TLR-4 [23]

 Non-T-cell cytotoxicity associated with hepatocyte damage [41]  CD4 T-cells from HLA-DRB1*0701þ patients proliferate and produce IFN-g in response to 7 CYP2D6 antigenic sequences [4]  CYP2D6-specific CD8 T-cells produce IFN-g and exert cytotoxicity in response to 5 CYP2D6 antigenic sequences [29]  CYP2D6245–254 is the HLA-A2-restricted sequence most effective at inducing CD8 T-cell effector functions [29]

SLA: soluble liver antigen; CYP2D6: cytochrome P4502D6; TCR: T-cell receptor; ASGPR: asialoglycoprotein receptor; ADH: alcohol dehydrogenase; IFN-g: interferon-gamma; TNF-a: tumour necrosis factor alpha; interleukin 10: IL-10.

3.5.1. Autoreactive CD4 T-cells T-cell ligands have been mainly studied in AIH type 2, where the autoantigen is known. The observation that autoantibodies to CYP2D6 belong to the IgG isotype, implicating a CD4-dependent class switch, and that the majority of T-cells infiltrating the liver in AIH are CD4 lymphocytes has led to studies investigating CD4 T-cell immune response against CYP2D6 in LKM-1 positive patients. CYP2D6262–285-specific T-cell clones generated from liver tissue and peripheral blood express a TH1 CD4þ phenotype [53,54]. At variance with these studies, which focused on a short antigenic sequence of CYP2D6, a systematic approach based on the construction of overlapping peptides covering the whole CYP2D6 molecule was recently adopted to define the specificity of ex vivo CYP2D6 reactive T-cells in patients with AIH type 2 [4]. This study showed that T-cells from patients positive for the predisposing HLA allele DRB1*0701 recognise in a proliferation assay seven regions of CYP2D6, four of which are also partially recognized by T-cells of DRB1*0701 negative patients. While distinct peptides induce production of IFN-g, IL-4 or IL-10, peptides that induced IFN-g and proliferative responses overlap. There was also an overlap between sequences inducing Tand B-cell responses. The number of epitopes recognized and the quantity of cytokine produced by T-cells are directly correlated to biochemical and histological markers of disease activity. These results indicate that the T-cell response to CYP2D6 in AIH type 2 is polyclonal, involves multiple effector types targeting different epitopes, and is associated with hepatocyte damage [4].

promoting the activation of different arms of the immune system. This region could therefore be an appropriate candidate for potential antigen-specific immune intervention.

3.5.2. Autoreactive CD8 T-cells In the early 1990s CD8 T-cell clones specific for ASGPR were described in patients with AIH [51]. Recent studies have identified CYP2D6-specific CD8 T-cells capable of secreting IFN-g and of exerting cytotoxicity after recognition of CYP2D6 epitopic sequences in an HLA class I restricted fashion [29]. The possibility that CYP2D6-specific CD8 T-cell immune responses are directly involved in the liver damage is suggested by the association of their strength with indices of disease activity and by their presence within the portal tract cellular infiltrate [29]. The CYP2D6245–254 sequence, which flanks a major B-cell epitope (CYP2D6254–271) [55] and overlaps with CYP2D6225–260, a T-cell epitope able to induce CD4 T-cell proliferation and IFN-g production [4], was identified as the HLA-A2-restricted sequence most effective at inducing CD8 T-cell functions [29]. The co-localisation of B, CD4 and CD8 T-cell epitopes suggests functional interrelations between immune responses to this particular region. B-cells can function as antigenpresenting cells to CD4 T-helper lymphocytes that in turn provide help to cytotoxic T-lymphocytes. The fact that B and T-cell immune responses converge on the same antigenic region indicates that this part of the CYP2D6 molecule may play a key role in the initiation and perpetuation of the autoimmune process in AIH type 2, by

4. Animal models

3.5.3. Monocytes In addition to CD4 and CD8 T-lymphocytes, monocytes/macrophages represent a major component of the portal/periportal cellular infiltrate in AIH. A recent study has demonstrated that compared to health, peripheral blood monocytes, isolated from children with AIH, have a more vigorous spontaneous migration, which cannot be further augmented by exposure to migrationinducing stimuli [23]. In addition, monocytes in AIH are skewed towards a pro-inflammatory phenotype, as indicated by the higher TNF-a over IL-10 production and by the elevated level of expression of Toll-like-receptor-4 (TLR-4), whose engagement is key to the initiation of adaptive immune responses. The finding of a marked monocyte activation during active disease, a time when also CD4 and CD8 T-cell autoimmune responses are at their highest in terms of proliferation and IFN-g production, suggests a monocyte participation in the pathogenesis of liver damage in AIH, possibly promoted by autoreactive cells belonging to the adaptive arm of the immune system [23]. Taken together, the data presented above indicate an involvement of both humoral and cellular immune responses in the pathogenesis of AIH, involvement which is likely to be permitted by a failure of immune homeostatic processes.

Research on the pathogenesis of AIH has been hampered by the lack of animal models reproducing faithfully the human condition. The ideal model for AIH should have a well-defined initiating event followed by chronic inflammation leading to fibrosis. Recently, researchers have focused on animal models of AIH type 2, since in this condition the autoantigen is well-defined. The model produced by the group of Alvarez [56] is based on immunizing every two weeks for three times C57BL/6 female mice with a plasmid containing the antigenic region of human CYP2D6, the target of antiLKM-1, and formimino-transferase cyclodeaminase, the target of anti-liver cytosol-1, an additional marker for AIH type 2 [57], together with the murine end terminal region of CTLA-4. The latter was added to facilitate antigen uptake by antigen-presenting cells. In a parallel set of experiments a plasmid containing the DNA encoding IL-12, a TH1 skewing pro-inflammatory cytokine, was also used. When autoantigens and IL-12 were used to break tolerance, antigen-specific autoantibodies were produced, a relatively modest elevation of transaminase levels at 4 and 7 months was observed, and a portal and periportal inflammatory infiltrate composed of CD4 and CD8 T-cells and, to a lesser extent, B-cells was

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demonstrated 8–10 months after the third immunization. When the same immunization protocol was used in different mouse strains, either a mild hepatitis or no inflammatory changes were observed indicating the importance of a specific genetic background. Another model of AIH type 2 uses CYP2D6 transgenic mice and aims at breaking tolerance with an Adenovirus-CYP2D6 vector [58]. While focal hepatocyte necrosis was seen in both mice treated with the Adenovirus-CYP2D6 vector and control mice treated with Adenovirus alone, only the former developed chronic histological changes, including fibrosis, reminiscent of AIH. The hepatic lesion was associated to a specific immune response to an immunodominant region of CYP2D6 and a cytotoxic T-cell response to Adenovirus-CYP2D6 vector infected target cells. Though these two experimental approaches provide useful information on the possible pathogenic mechanisms leading to AIH type 2, a model mimicking closely AIH in humans is still missing. Additional insight into the role of antigen-specific T-cells in the induction of acute disease and its perpetuation has been recently provided by a transgenic mouse model, which expresses chicken ovalbumin (OVA) on the hepatocyte surface [59]. Repeated injections (every 2 weeks for a total of 3 injections) of OVA-specific T-cells resulted in chronic hepatitis characterized by lobular and portal inflammation. Hepatic damage was determined by CD8 OVAspecific T-cells and was enhanced by OVA-specific CD4 T-cell help [59]. In contrast to this finding, supporting a prominent role for CD8 T-cells in AIH pathogenesis, Robinson et al. reported that in TGF-b/ mice, which develop fulminant AIH, liver damage is driven by CD4 T-cell production of IFN-g, while it is independent on CD8 T-cells, thus indicating that a differential participation of immune-mediated mechanism may determine a different disease outcome [60]. References [1] Donaldson PT. Genetics in autoimmune hepatitis. Semin Liver Dis 2002;22:353–64. [2] Donaldson PT. Genetics of autoimmune and viral liver diseases; understanding the issues. J Hepatol 2004;41:327–32. [3] Czaja AJ, Donaldson PT. Genetic susceptibilities for immune expression and liver cell injury in autoimmune hepatitis. Immunol Rev 2000;174:250–9. [4] Ma Y, Bogdanos DP. Polyclonal T-cell responses to cytochrome P450IID6 are associated with disease activity in autoimmune hepatitis type 2. Gastroenterology 2006;130:868–82. [5] Duarte-Rey C, Pardo AL. HLA class II association with autoimmune hepatitis in Latin America: a meta-analysis. Autoimmun Rev 2009;8:325–31. [6] Agarwal K, Czaja AJ. Cytotoxic T lymphocyte antigen-4 (CTLA-4) gene polymorphisms and susceptibility to type 1 autoimmune hepatitis. Hepatology 2000;31:49–53. [7] Bittencourt PL, Palacios SA. Cytotoxic T lymphocyte antigen-4 gene polymorphisms do not confer susceptibility to autoimmune hepatitis types 1 and 2 in Brazil. Am J Gastroenterol 2003;98:1616–20. [8] Czaja AJ, Cookson S. Cytokine polymorphisms associated with clinical features and treatment outcome in type 1 autoimmune hepatitis. Gastroenterology 1999;117:645–52. [9] Yoshizawa K, Ota M. Genetic analysis of the HLA region of Japanese patients with type 1 autoimmune hepatitis. J Hepatol 2005;42:578–84. [10] Yokosawa S, Yoshizawa K. A genomewide DNA microsatellite association study of Japanese patients with autoimmune hepatitis type 1. Hepatology 2007;45:384–90. [11] Simmonds MJ, Gough SC. Genetic insights into disease mechanisms of autoimmunity. Br Med Bull 2004;71:93–113. [12] Mathis D, Benoist C. Aire. Annu Rev Immunol 2009;27:287–312. [13] Lankisch TO, Strassburg CP. Detection of autoimmune regulator gene mutations in children with type 2 autoimmune hepatitis and extrahepatic immune-mediated diseases. J Pediatr 2005;146:839–42. [14] Lankisch TO, Mourier O. AIRE gene analysis in children with autoimmune hepatitis type I or II. J Pediatr Gastroenterol Nutr 2009;48:498–500. [15] Nouri-Aria KT, Hegarty JE. Effect of corticosteroids on suppressor-cell activity in ‘‘autoimmune’’ and viral chronic active hepatitis. N Engl J Med 1982;307:1301–4. [16] Vento S, Hegarty JE. Antigen specific suppressor cell function in autoimmune chronic active hepatitis. Lancet 1984;1:1200–4. [17] Longhi MS, Ma Y. Impairment of CD4(þ)CD25(þ) regulatory T-cells in autoimmune liver disease. J Hepatol 2004;41:31–7. [18] Longhi MS, Ma Y. Effect of CD4þ CD25þ regulatory T-cells on CD8 T-cell function in patients with autoimmune hepatitis. J Autoimmun 2005;25:63–71.

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