Autoimmune manifestations in viral hepatitis | SpringerLink

Semin Immunopathol (2013) 35:73–85 DOI 10.1007/s00281-012-0328-6

REVIEW

Autoimmune manifestations in viral hepatitis Diego Vergani & Giorgina Mieli-Vergani

Received: 3 February 2012 / Accepted: 1 July 2012 / Published online: 28 July 2012 # Springer-Verlag 2012

Abstract Infections by the viruses responsible for hepatitis B, C and D are accompanied by a number of immunopathological manifestations. A link between infection and autoimmunity is particularly well documented for the hepatitis C virus. Immunopathological manifestations range from production of autoantibodies to overt autoimmune disease, including thyroiditis and autoimmune hepatitis, and to immune-complex-mediated disorders, including cryoglobulinaemia, glomerulonephritis and vasculitis. Several of these manifestations improve with successful antiviral treatment, directly incriminating the virus in their pathogenesis. Mechanisms considered responsible for hepatitis virus-related immunopathology, including molecular mimicry, impairment of regulatory T cells and activation of B lymphocytes, will be examined in this review.

this review, autoimmune features of viral hepatitis B, C and D, ranging from autoantibody seropositivity to open autoimmune disease, will be examined and a link between the virus infections and autoimmunity will be sought. As immunopathological manifestations are especially frequent in hepatitis C virus (HCV) infection, HCV will be considered first.

Keywords Hepatitis C . Hepatitis B . Hepatitis D . Autoimmunity . Immune complex disease . Molecular mimicry . Regulatory T cells . B lymphocyte activation

Autoantibodies

Introduction In the 1990s, Rolf Zinkernagel stated that ‘… an autoimmune disease is a viral disease in which the virus is unknown’ [1]. In This article is published as part of the Special Issue on Immunopathology of viral hepatitis D. Vergani : G. Mieli-Vergani Institute of Liver Studies and Paediatric Liver, GI & Nutrition Centre, King’s College Hospital, King’s College London School of Medicine, Denmark Hill, London SE5 9RS, UK D. Vergani (*) Institute of Liver Studies, King’s College Hospital, Denmark Hill, London SE5 9RS, UK e-mail: [email protected]

Hepatitis C virus From its discovery in 1989, HCV has been linked to a variety of immunopathological phenomena, including cryoglobulinaemia, B cell lymphoma, autoantibody production and overt autoimmune disease [2–4].

Non-organ-specific autoantibodies have been consistently described in chronic HCV infection, with prevalence as high as 70 % [5]. The most frequent autoantibody encountered is anti-smooth muscle antibody (SMA; up to 66 %), followed by anti-nuclear antibody (ANA; up to 41 %) and, in a minority of cases, anti-liver kidney microsome type 1 (anti-LKM1; 1–11 %) antibody [5–7]. The susceptibility to develop non-organ-specific autoantibodies is related to the genetic background that predisposes to autoimmune hepatitis (AIH). In particular, HLA A1-B8-DRB1*0301 and DRB1* 0401 are associated to the development of ANA [8, 9], while HLA-DRB1*0701 and DQB1*0201 are significantly more frequent in anti-LKM1-positive than in antiLKM1-negative HCV patients [9]. The immunofluorescent patterns of SMA and ANA, determined on rodent tissue sections [10] and HEp-2 cells [11], respectively, tend to differ from those typically reported in AIH [12]. While in AIH type 1 SMA gives the ‘actin’ pattern, staining arterial vessels (V), renal glomeruli (G) and tubules (T), SMA in HCV infection is usually limited to the V pattern

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(Fig. 1a). ANA, which typically has a homogeneous appearance in AIH type 1, usually gives a speckled staining in HCV infection (Fig. 1b). In contrast, the immunofluorescent pattern of anti-LKM1 (bright staining of liver cell cytoplasm and P3 portion of renal tubules) is indistinguishable from that of type 2 AIH [11, 12] (Fig. 2a). Reactivity to liver cytosol type I (antiLC1) (Fig. 2b), an autoantibody also present in AIH type 2, has been reported in a small proportion of patients with chronic HCV infection, frequently in association with anti-LKM1, which, when the two autoantibodies co-occur, masks the antiLC1 immunofluorescent pattern making its detection difficult [13]. The prevalence of the anti-LC1 reactivity in HCV infection depends on the system used for its assessment, being 12 % by western blot [14] and 5 % by commercial ELISA [15]. Other autoantibodies present in patients with HCV infection include anti-thyroid peroxidase antibodies (anti-TPO) and, less frequently, anti-gastric parietal cell, anti-mitochondrial and antineutrophil cytoplasmic antibodies [5–7, 16–20]. Fig. 1 Anti-smooth muscle (SMA) and anti-nuclear (ANA) antibodies in chronic viral hepatitis. The immunofluorescent patterns of SMA and ANA in chronic hepatitis C, B and D are similar, but differ from those detected in autoimmune hepatitis type 1. a On a rodent kidney section, the staining of SMA in viral hepatitis is confined to vessels (V; left), while SMA in autoimmune hepatitis also stains the glomeruli (VG; right). b On Hep2 cells, the pattern of ANA in viral hepatitis is typically speckled (left), while in autoimmune hepatitis it is homogeneous (right)

Semin Immunopathol (2013) 35:73–85

The presence of ANA, SMA and anti-LKM1 in chronic HCV infection is unlikely to be an epiphenomenon, as it is associated with presence of liver damage. During the screening of an unselected Italian population of 7,000 subjects [21] for clinical and laboratory evidence of liver disease (Dionysos study [21]), 226 were found to be positive for HCV markers [22]. Autoantibodies were detected in 25 % of them, a prevalence much higher than that observed in 226 demographically matched uninfected individuals and in 78 hepatitis B surface antigen (HBsAg)-positive subjects within the same study group [6]. In the HCV-positive cohort, the presence of autoantibodies was significantly associated with clinical and biochemical evidence of liver disease, suggesting a connection between the two. Of note, several reports describe adverse reactions to interferon treatment in autoantibody-positive HCV patients [23–26], but invariably the autoantibody associated with these reactions is anti-LKM1. Anti-LKM1 appears to act as


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disease. In the study of Ferri et al., hepatitic flares during antiviral treatment occurred only in patients positive for antiLKM1, but after a short corticosteroid course, these patients eventually responded to antiviral treatment similarly to those who were anti-LKM1 negative. From several reports [27–29], it appears that anti-LKM1positive patients fall into two distinct groups according to their HCV markers. Anti-LKM1-positive subjects without HCV markers probably represent a true autoimmune group: They tend to be children or young adults, female, with either a family history of autoimmune disease or associated autoimmune disorders and respond to immunosuppressive treatment. Anti-LKM1-positive patients with markers of HCV infection are usually older, male, without a clear association with other autoimmune manifestations and respond better to antiviral than immunosuppressive treatment. Studies comparing sera from HCV-positive and HCV-negative patients with identical LKM1 immunofluorescent pattern and similar titres have shown that LKM1-positive/anti-HCV-positive sera, mainly from adult patients, react significantly less frequently with prokaryotically expressed cytochrome P4502D6 (CYP2D6), the target of anti-LKM1 in autoimmune hepatitis, than antiLKM1-positive/HCV-negative sera, mainly from children [30]. When a eukaryotically expressed CYP2D6 was used, a similar frequency of reactivity was observed in anti-LKM1positive/HCV-positive and anti-LKM1-positive/HCV-negative patients, though HCV-negative patients recognised more frequently the sequence CYP2D6267–337 [31]. Anti-LKM1 reactivity in HCV infection, therefore, despite being also directed to CYP2D6, has a pattern of epitope recognition that partially differs from that of patients with AIH type 2. The possible relationship between HCV infection and development of AIH type 2 will be discussed below within the “Mechanisms” section. Fig. 2 Anti-liver kidney microsomal type 1 (anti-LKM1) and anti liver cytosol type 1 (anti-LC1) antibodies in chronic hepatitis C virus (HCV) infection. The immunofluorescent patterns of anti-LKM1 and anti-LC1 in HCV infection are similar to those of autoimmune hepatitis type 2. AntiLKM1 stains the cytoplasm of hepatocytes and the proximal renal tubules (a), while anti-LC1 stains the hepatocytes sparing those around the central lobular vein (b) (rat renal and liver tissues). Anti-LKM1 and anti-LC1 are not associated with chronic hepatitis B or D viral infections

Immune-complex-mediated pathology Chronic HCV infection is associated with extrahepatic disorders, in which HCV containing immune complexes are likely to play a major pathogenic role. Cryoglobulinaemia

‘danger’ marker whatever its titre and should be measured in all HCV patients, especially those undergoing interferon treatments. In the paediatric study by Gregorio et al. [5], four children with HCV infection had to suspend interferon treatment because of marked transaminase elevation: Three were anti-LKM1 positive and two at a titre as low as 1/10. In that series, there was only one additional LKM1-positive patient who received interferon without an elevation of transaminases. Cassani et al. [18] and, more recently Ferri et al. [26], have shown that the presence of anti-LKM1 in HCV infection is associated with a greater histological severity of liver

Cryoglobulins are immunoglobulins (Ig) that become insoluble below 37 °C and dissolve again at 37 °C or above. Cryoglobulins are classified in three types: In type I, the cryocrit contains a monoclonal Ig (or light chain); in type II, a polyclonal IgG and a monoclonal IgM with reactivity for IgG (rheumatoid factor, RF) and in type III, both IgG and IgM are polyclonal, IgM still having RF activity. Type II and type III are defined as mixed cryoglobulinaemias and are associated with HCV infection [3, 32, 33], their prevalence ranging from 19 to 65 % among HCV-infected patients [3, 33–35].

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Such variability in prevalence is probably due to technical reasons in the detection of cryoglobulins and to a referral bias, the highest prevalence reported coming from a centre specialized in HCV-related extrahepatic manifestations [35]. The cryoglobulinaemic syndrome comprises purpura, weakness and arthralgia (Meltzer’s triad), but often cryoglobulinaemia is clinically silent, symptoms usually arising only when the cryocrit is abundant. Additional manifestations of cryoglobulinaemia include Raynaud’s phenomenon, peripheral neuropathy, sicca syndrome and membranous proliferative glomerulonephritis. In cryoglobulinaemic patients chronically infected by HCV, both HCV RNA and anti-HCV antibody have been detected in the cryocrit [36], and HCV has been demonstrated in the cutaneous vasculitic lesions of patients with type II cryoglobulinaemia [37], indicating a direct involvement of the virus in these manifestations. The question arises as to why only some patients with chronic HCV infection have cryoglobulinaemia. There are several possible explanations: The viral load can vary by several orders of magnitude; the host anti-viral immune response can be more or less vigorous, with antibodies being directed to different components of the virus and being of different affinity; genetic factors pertaining to the virus or the host may also be involved. Willems et al. found HCV genotype I in virtually all patients with mixed cryoglobulinaemia they studied [38], though the same genotype was also present in a high proportion of HCV patients without cryoglobulinaemia. Within an Italian population, no association with a specific HCV genotype was identified [39], while in a comparative study between Japan and Egypt, HCV genotype 1b was reported to predispose to cryoglobulinaemia more than genotype 4 [40]. Sustained clearance of HCV following anti-viral treatment with alpha-interferon and ribavirin leads to a complete resolution of cryoglobulinaemia, reiterating the direct role of the virus in its causation [41–47]. Eradication of HCV by antiviral therapy is therefore the treatment of choice for mixed cryoglobulinaemia secondary to this infection, but in patients who do not respond to antiviral treatment or suffer from severe vasculitis and/or skin ulcers, peripheral neuropathy or glomerulonephritis, immunosuppressive therapies are employed, including anti-CD20 monoclonal antibody (rituximab) [48, 49] Glomerulonephritis Glomerulonephritis is the archetype of immune-complexmediated disorders, where immune deposits containing immunoglobulin complement components and antigens are detectable in the glomeruli. Several reports link HCV to membranous proliferative glomerulonephritis, generally in association with mixed cryoglobulinaemia [50–54]. In areas endemic for HCV infection, the proportion of HCV positivity among patients with glomerulonephritis and cryoglobulinaemia is as high as 85–100 % [55–58]. The demonstration of


HCV in the immunocomplexes deposited in the glomeruli [59] and the simultaneous improvement of the renal and hepatic disease with alpha-interferon treatment incriminate HCV as an aetiological agent of glomerulonephritis [60]. Sjögren’s syndrome A viral aetiology has been repeatedly suggested for this autoimmune condition affecting predominantly lacrimal and salivary glands and leading to the so-called sicca syndrome. The presence of xerostomia, sialadenitis, abnormal salivary flow rates and abnormal Schirmer test has been repeatedly reported in patients with HCV infection, reaching frequencies of over 50 %, although the classical serology of Sjögren’s syndrome— antibodies to Ro/SSA or La/SSB—is found in only 1 % [61, 62]. The presence of HCV in saliva and in the mononuclear cells of infected patients supports the possibility of a pathogenic role for HCV in Sjögren’s syndrome. Autoimmune endocrine disorders Thyroid disease Thyroid involvement is the most common endocrine manifestation in chronic HCV infection. Anti-thyroid antibodies are variably reported in 2–48 % of patients [63–66], while subclinical hypothyroidism is described in 2–9 % [63–67]. The prevalence of thyroid autoimmune disorders is higher in HCVinfected patients than in the general population and in hepatitis B virus (HBV)/hepatitis D virus (HDV)-infected patients [63, 67, 68]. The variable reported prevalence of thyroid involvement in HCV infection may derive from different genetic susceptibility or from environmental factors, such as exposure to iodine [69, 70]. Thyroid dysfunction can precede antiviral treatment, but frequently emerges or worsens during interferonbased therapy, particularly in females with anti-TPO antibodies without clinical manifestations before treatment. This suggests that interferon, a drug known to favour or exacerbate autoimmunity, enhances the thyroid autoimmune process. It is therefore important to assess regularly thyroid function in patients with chronic HCV infection before and during treatment to take appropriate action should problems arise. Hypothyroidism is the prevalent manifestation, hyperthyroidism being similarly reported in HCV-infected and control populations [68, 71, 72]. Diabetes A high prevalence of diabetes mellitus type 2 has been reported in patients with chronic HCV infection and ascribed to direct viral infection of the beta-cells [3, 73]. Type 1 diabetes is not more prevalent in HCV-positive patients than in the uninfected population, but interestingly, treatment with interferon for HCV infection has been anecdotally


reported to trigger autoimmunity against pancreatic islets and development of type 1 diabetes [31, 74].

Hepatitis B virus HBV infection has also been associated to a variety of immunopathological manifestations [75], including circulating nonorgan-specific autoantibodies, membranous/membranous proliferative glomerulonephritis [76], mixed cryoglobulinaemia [77] and polyarteritis nodosa [78]. SMA and ANA autoantibodies, with an immunofluorescent pattern similar to that of HCV infection, have been documented in chronic HBV infection irrespective of interferon treatment, but their prevalence is usually lower than that observed in HCV-infected patients [6, 79]. Anti-LKM1 reactivity is extremely rare. Chronic HBV infection has been reported in essential mixed cryoglobulinaemia [80], though much less frequently than in HCV infection [77, 81], and is associated to membranous or membranoproliferative glomerulonephritis, where the granular deposits present in the glomeruli contain HBV antigens, in particular hepatitis B e antigen (HBeAg) [82], in addition to anti-viral antibodies [83]. This finding and the observation of renal disease remission following inhibition of viral replication with interferon or nucleot(s)ide inhibitor treatment supports the pathogenic role of HBV [4, 84, 85]. About a third of patients with polyarteritis nodosa are infected by HBV. The vasculitic lesions usually appear during primary HBV infection and are related to the presence of HBeAg [86–88]. Anti-HBe seroconversion, either spontaneous or induced by antiviral treatment, leads to the resolution of the vasculitic process [89–91]. Other cutaneous manifestations of HBV infection are the Gianotti–Crosti acrodermatitis, affecting mainly children [92], and an acute urticarial rash, the immune histology of which shows vasculitic features with deposition of complement factor C3, IgM and HBsAg [93].

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personal communication, data presented at the EASL monothematic conference on Delta Hepatitis, Istanbul, 24–26 September 2010). Despite its high specificity with HDV infection, no association has been documented with clinical or laboratory indices. Anti-LKM3 gives an immunofluorescent pattern characterised by liver and kidney staining only detectable on primate/ human tissues [96]. The target of anti-LKM3 has been identified as uridine diphosphate glucuronosyl transferase (UGT) [97] and shown by Bachrich et al. [98] to block UGT enzymatic activity. Its clinical relevance remains to be established. Wesierska-Gadek et al. have described autoantibodies to the nuclear membrane lamin C in a high proportion of patients with chronic HDV infection [99], a reactivity present also in AIH and primary biliary cirrhosis.

Mechanisms The mechanisms leading to extrahepatic immunopathological manifestations in chronic viral hepatitis are diverse and range from molecular mimicry to an imbalance of T effector cells and T regulatory cells (Treg) and to a direct effect of the virus on B lymphocytes. Molecular mimicry Molecular mimicry is an extensive homology between microorganisms (bacteria, viruses or parasites) and selfantigens, probably representing a defence mechanism for infecting microorganisms to avoid the anti-microbial immune response [100]. However, an immune response initially directed against an exogenous antigen and crossreacting with homologous host sequences can lead to autoreactivity and autoimmune disease [101]. Following the observation that homologous sequences are not exclusive to one single exogenous and endogenous pair of mimicking

Hepatitis D virus ANA and SMA are present also in HDV infection, with a prevalence and immunofluorescent pattern similar to that found in HBV infection (Lenzi, personal communication, data presented at the EASL monothematic conference on Delta Hepatitis, Istanbul, 24–26 September 2010; Mytilinaiou, unpublished data). Peculiar to HDV infection is the presence of anti-basal cell layer (BCLA) (Fig. 3) and anti-liver kidney microsome type 3 (LKM3) autoantibodies. Anti-BCLA, detected by immunofluorescence on rat forestomach [94], is closely associated with HDV infection, but its prevalence varies greatly between reports, being as high as 60–90 % in some [95] and around 15 % in others (Lenzi,

Fig. 3 Basal cell layer antibody (BCLA). BCLA stains the basal cell layer of stratified epithelia (rat forestomach or primate oesophagus). BCLA is present in a variable proportion of patients with HDV infection but is absent in HBV mono-infection or in HCV infection (picture kindly provided by Professor Marco Lenzi)

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antigens but can be shared by several pathogens and one self-molecule, a multiple hit theory has been proposed: The contact with the first exogenous epitope mimicking a selfantigen would prime the immune system (‘original antigenic sin’) leading over time to expanding self-reactivity dependent on subsequent encounters with pathogens sharing similar sequences with the original self-antigen [102]. The initial cross-recognition of an exogenous antigen and self can progress to inter and intra-molecular epitope spreading, i.e. spreading of the immune response from an epitope on one molecule to an unrelated epitope on the same molecule or on a different, contiguous, molecule. The amplified immune response can include self-epitopes distinct from those that initiated the process. Once the process is established, the initial antigen may no longer be required for the maintenance of the autoimmune response [103]. Molecular mimicry between HCV and HBV components and self-antigens may explain the presence of most of the autoimmune features accompanying HCV and HBV chronic infection [104–106]. HCV Scanning of protein databases has revealed regional similarities between HCV proteins and putative antigenic targets of ANA and SMA [106]. The HCV polyprotein shares six to nine amino acid sequences in common with antigenic targets of ANA (matrin, histone, replication protein) and SMA (vimentin, myosin, smoothelin). Double reactivity to the HCV polyprotein and matrin, histone, replication protein, vimentin, myosin or smoothelin was observed in 15–53 % of HCVpositive patients, but in only 3 % of patients with non-viral chronic liver disease and in none of healthy controls [106]. Double reactivity to smooth muscle and HCV peptide antigens correlated with SMA positivity by indirect immunofluorescence. Eighty-seven percent of patients double reactive to myosin1035–1054 and its HCV homologue recognised whole myosin by immunoblot [106]. That molecular mimicry may be involved in the production of anti-LKM1 antibody—which targets CYP2D6—in patients with chronic HCV infection as first suggested by Manns et al. [107], who noted that the sequence CYP2D6252–271 shares amino acid homology with sequences of the non-structural 5B and the envelope 1 proteins of the HCV polyprotein and proposed that anti-LKM1 reactivity in HCV infection results from cross-reactivity between virus and self. This hypothesis was later supported by experimental data, demonstrating humoural cross-reactivity between HCV and CYP2D6 [108]. This cross-reactivity, however, is dependent on a specific genetic background, i.e. possession of HLA B51 [108]. Cross-reactivity is not confined to the humoural arm of the immune system, as the sequence CYP2D6313–332, sharing extensive homology with HCV794–801, is recognised by CD4 T cells from patients with chronic HCV infection [109]. As mentioned earlier, positivity for anti-LKM1 in HCV infection is associated with more severe histological changes and with


hepatitic flares during interferon treatment, suggesting that it might be directly involved in liver damage. For anti-LKM1 to be pathogenically relevant, its main target, CYP2D6, should be easily accessible to the autoantibody. The demonstration by Muratori et al. [110] that CYP2D6 is present not only in the endoplasmic reticulum but also on the liver cell membrane makes it an ideal target for an autoantibodymediated attack. Moreover, as previously shown in vitro, a recent paper shows an 80 % reduction of CYP2D6 metabolic activity in vivo in patients with chronic HCV infection and anti-LKM-1 antibodies [111]. The possible involvement of HCV in causing autoimmunity is supported by an informative clinical case [112]. A child started producing anti-LKM1 antibody 2 weeks after becoming infected by HCV following liver transplantation for endstage liver disease due to alpha 1 anti-trypsin deficiency. A temporal relationship between HCV infection and development of anti-LKM1 was noted, suggesting a causal relationship between the two. The production of this autoantibody showed the characteristics of a physiological immune response, anti-LKM1 belonging initially solely to the IgM isotype, consisting of IgM and IgG for a brief subsequent period and then becoming exclusively IgG. Of note, 8 years after developing anti-LKM1, when the patient had become HCV PCR and anti-HCV negative, she developed graft dysfunction associated with anti-LKM1 positivity and severe interface hepatitis on liver biopsy leading to the diagnosis of de novo AIH type 2, which responded well to the standard treatment for AIH, i.e. prednisolone and azathioprine [113]. Review of her clinical history revealed that she had been exposed over the years to Herpes simplex type 1, cytomegalovirus and Epstein–Barr virus, microorganisms which share homologous sequences with HCV and CYP2D6, suggesting that her autoimmune liver disease may be the result of multiple exposures to self-mimicking microbial sequences. Interestingly, the girl is HLA B51 positive. The mechanisms leading to the development of anti-thyroid antibodies and thyroid disorders during chronic HCV infection, especially during interferon-based antiviral therapy, are only partially understood. A study in a large HCV patient cohort has reported a close association between anti-thyroid and anti-LKM1 antibodies [104]. In that study, thyroid peroxidase (a key autoantigen in autoimmune thyroid disease), CYP2D6 and HCV polyprotein were shown to share three distinct amino acid homologies, patients with the triple reactivity being more likely to have autoimmune thyroid disease. Anti-LKM1/HCV-positive patients were found to be 14 times more likely to develop autoimmune thyroiditis during interferon treatment than anti-LKM1-negative patients. A role for molecular mimicry has been suggested by Hartmann et al. [114] also in the pathogenesis of HCVrelated cryoglobulinaemia. These authors found that in some cryoglobulinaemic HCV-positive patients, not only the IgG


but also the IgM component of the cryocrit reacts with HCV proteins. In view of the fact that the IgM component of the cryoprecipitate has RF activity, i.e. it reacts with IgG, the authors performed a computer-assisted search of sequence homologies, finding the motif EGLGWAGWL to be shared in common between the heavy chain of IgG and the core protein of HCV. HBV Scanning of protein databases has revealed regional similarities also between HBV proteins and putative antigenic targets of ANA and SMA [105]. HBV-DNA polymerase (HBV-pol) shares six to nine amino acid sequences in common with antigenic targets of ANA [MHC transactivator (TA), nuclear core protein (NCP), nuclear mitotic apparatus (NUMA), polymyositis sclerosis antigen (Pm-scl)] and SMA (myosin, caldesmon). Double reactivity to HBV-pol peptides and TA, NCP, NUMA, PM-Scl, caldesmon and myosin homologues was present in 26–40 % of HBVpositive patients, but absent in non-viral chronic liver disease and health. Double reactivity to myosin or caldesmon peptides and their HBV-pol homologues was associated with SMA positivity by immunofluorescence. HBVpositive sera double reactive for myosin or caldesmon and their homologous HBV-pol peptides also reacted with the native proteins on immunoblot [105]. Regulatory T cells

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obtaining an increase in Treg number and a reduction in cryoglobulinaemia in nine patients and improvement of vasculitis in eight. This important pilot clinical study suggests a direct link between defective Treg and HVC-related cryoglobulinaemia/vasculitis. HBV In HBV infection, similarly to HCV, Tregs have been reported to be increased in patients with chronic infection and suggested to favour viral persistence, though this view is not unanimously held [125]. No direct link between Treg number and/or function and HBV-related immunopathological manifestations has been reported. B lymphocytes B lymphocytes play an important role in the development of immunopathological manifestations of chronic viral hepatitis, as clearly documented in chronic HCV infection. B cell clonal expansion Type II mixed cryoglobulinaemia syndrome, so commonly associated with chronic HCV infection, is considered a precursor of B cell non-Hodgkin lymphoma (NHL). A monoclonal lymphoproliferative infiltrate of uncertain significance has been described in the liver and bone marrow of subjects with clinical and laboratory features of cryoglobulinaemia type II [126]. Several studies show that B cell clonal expansion (in particular of RF B cells) underlies mixed cryoglobulinaemia [127], that this

The function of regulatory cells is to inhibit immune responses and prevent autoimmunity. The regulatory population most extensively characterised is a small subset of CD4+ T cells identified by their constitutive expression of CD25, the alpha chain of interleukin 2 (IL2) receptor and high levels of the lineage-specific transcription factor FOXP3 (CD4+CD25+ Treg) [115, 116]. Tregs are defective in number and/or function in several autoimmune conditions [117, 118], while their number is increased during infection and neoplasms [119]. HCV Increased numbers of Tregs are present in patients with chronic HCV infection and in those with acute infection that becomes chronic [120–122]. Their increase, probably aiming at preventing tissue damage due to the action of effector cells fighting the infection, would also prevent efficient clearance of the virus. In contrast, patients chronically infected with HCV who develop symptomatic mixed cryoglobulinaemia have a reduction in their peripheral Treg number [123], suggesting that lack of immunoregulatory function favours immunopathological manifestations. Exploiting the low threshold of Treg in response to IL2, Saadoun et al. [124] have recently treated ten patients with HCV-induced vasculitis, refractory to conventional antiviral therapy and anti-CD20 monoclonal antibody, with repeated courses of low dose IL2

Fig. 4 Hepatitis C virus (HCV) activation of B lymphocytes. B lymphocytes have a receptor for HCV, CD81, a tetraspanin that has the envelope 2 (E2) protein of the virus as a natural ligand. Closely associated to CD81 are the B lymphocyte specific proteins CD21 and CD19. The first is the receptor for the complement C3d fragment, while the second transduces activation signals into the cell. CD19 also binds B-lymphocyte activating factor (BAFF), the levels of which are elevated in HCV infection. When HCV coated by C3d engages CD81 and BAFF binds CD19, the B cell threshold for polyclonal activation is lowered, leading to production of autoantibodies and cryoglobulins

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condition is associated with Bcl-2/JH—a B lymphocytespecific chromosomal rearrangement—and that type II mixed cryoglobulinaemia evolves into a frank B cell NHL in approximately 8–10 % of cases [127, 128]. Development of NHL in HCV chronically infected patients has been proposed to derive either from direct infection of B lymphocytes [129–131] or from their chronic antigenic stimulation [132, 133]. B cell activation A 100-fold reduction of the B lymphocyte activation threshold is present in patients chronically infected with HCV. This reduction in activation threshold is due to the binding of HCV to a number of molecules on the surface of B lymphocytes: HCV is recognised by the B cell receptor for antigen, the HCV envelope 2 protein binds the tetraspanin CD81 [134] and following complement activation, HCV coated by the complement fragment C3d engages CD21, the C3d receptor, which is associated in a co-stimulatory complex with CD19 [135] (Fig. 4). Moreover, B lymphocyte activating factor (BAFF), a member of the TNF cytokine family supporting B cell proliferation, is increased in HCV infection [136]. BAFF binds CD19, further lowering the threshold for B cell activation, and its elevation in HCV infection is associated with clinical and laboratory features of autoimmunity [137, 138]. The nonantigen-specific and polyclonal activation resulting from the engagement of co-stimulatory molecules on the surface of B lymphocytes may be responsible for the extrahepatic manifestations which characterise HCV infection, such as autoantibody and cryoglobulin production. Little is known about the effect of chronic HBV infection on B lymphocytes, though there is evidence that HBV is present in these cells [139]. A possible pathogenic link between HBV infection and B cell NHL has been reported, infected subjects being almost three times more likely to develop NHL than controls [140–143].

Conclusion Chronic infection with the hepatotropic viruses B, C and D is associated with diverse immunopathological manifestations, involving autoantibody production and immunecomplex-related pathologies. The link between these viruses and development of overt hepatic and extrahepatic autoimmune disease is less well-defined, patients who do develop overt autoimmune disease possessing a predisposing genetic background and/or having been exposed to cytokines favouring autoimmunity, such as alpha interferon. Going back to Rolf Zinkernagel’s statement mentioned at the beginning of this review, it is likely that further elucidation of the mechanisms linking hepatitis viruses and autoimmune


manifestations will help in understanding the process underpinning the loss of self tolerance.

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