a Institute of Liver Studies, King’s College London, Faculty of Life Sciences & Medicine, London, UKb Department of Gastroenterology, Centro Hospitalar São João and Faculty of Medicine, University of Porto, Porto, Portugalc Paediatric Liver, GI & Nutrition Centre, King’s College Hospital, London, UK
toinmune (HAI) es una hepatopatía inflamatoria progresiva y unaciencia hepática terminal. Su origen continúa siendo una incóg-
PALABRAS CLAVEHepatitisautoinmune;Patogenia;
La hepatitis autoinmune
Resumen La hepatitis aucausa importante de insufi
Please cite this article in press as: Liberal R, et al. Autoimmune hepatitis: From mechanisms to therapy. Rev Clin Esp.2016. http://dx.doi.org/10.1016/j.rce.2016.04.003
nita, si bien influyen en su evolución factores tanto genéticos como ambientales. El principal
∗ Corresponding author.E-mail address: [email protected] (D. Vergani).
utoimmune hepatitis (AIH) is an immune-mediated inflam-atory liver disease characterised serologically by hyper-
ammaglobulinaemia and the presence of non-organ andiver-specific autoantibodies, and histologically by a denseononuclear cell infiltrate in the portal tract.1 Two types
f AIH are recognised: AIH type 1 (AIH-1), defined by theresence of anti-smooth muscle (SMA) and/or anti-nuclearANA) antibodies; and AIH type 2 (AIH-2), defined by posi-ivity for anti-liver kidney microsomal type 1 (anti-LKM-1)nd/or anti-liver cytosol type 1 (anti-LC-1) antibodies.2,3
AIH occurs globally, affecting children and adults of allges and both sexes, although it is more commonly foundn females.4 There are no robust epidemiological data onIH. Available studies show an incidence of 1.5---2.0 caseser 100,000 people per year in Northern Europe.5,6 Preva-ence estimates range from 16.9 cases per 100,000 people inorthern Europe to 35.9 cases per 100,000 people in Alaskanatives.5,7 While AIH-1 affects both children and adults, AIH-
is mainly a paediatric condition.8 Female predominance is feature of both types.8
The mechanisms responsible for its development includeenetic predisposition to autoimmunity through possessionf specific human leucocyte antigen (HLA) alleles, immuneeactions to liver cell antigens, possibly triggered by a mech-nism of molecular mimicry, and impairment in immuneegulation.9
AIH has variable modes of presentation: it can presentnsidiously, with complications of portal hypertension, orith a clinical picture of acute hepatitis, or more rarelyith acute liver failure (ALF).4,10 In addition, variant forms
Please cite this article in press as: Liberal R, et al. Autoimmu2016. http://dx.doi.org/10.1016/j.rce.2016.04.003
f the disease, presenting with concomitant features ofrimary biliary cholangitis (PBC) or primary sclerosingholangitis (PSC) are increasingly recognised.11 AIH shoulde considered during the diagnostic workup of any increase
fwTf
n liver enzyme levels.12 A set of inclusion and exclusionriteria for the diagnosis of AIH have been established, andeanwhile revised, by the International Autoimmune Hep-
titis Group (IAIHG).13,14 Immunosuppressive therapy withteroids, with or without azathioprine, remains the mainstayf treatment, and should be instituted as soon as the diag-osis is made.1 Difficult-to-treat or non-responsive patientshould be treated with mycophenolate mofetil (MMF) or cal-ineurin inhibitors (CNI).1,4 Persistent failure to respond orack of adherence to treatment result in end-stage liver dis-ase. Patients with end-stage liver disease, and those withLF at diagnosis, will require liver transplantation (LT).15
This review aims to discuss the recent advances in thenderstanding of AIH pathogenesis, diagnosis, and treat-ent.
enetics
IH is a ‘‘complex trait’’ disease that does not follow aendelian pattern of inheritance. The mode of inheritancef a complex trait disorder is unknown, though it involvesne or more genes, operating alone or in concert, to increaser reduce the risk of the trait and interacting with environ-ental factors.Predisposition to AIH is linked to Major Histocompati-
ility Complex (MHC) class II genes, more specifically tohe Human Leucocyte Antigen (HLA)-DR locus, located onhe short arm of chromosome 6 --- which are involved inhe presentation of antigenic peptides to T-cells, and areherefore implicated in the initiation of an adaptive immuneesponse.16,17 In Europe and North America, the alleles con-
ne hepatitis: From mechanisms to therapy. Rev Clin Esp.
erring susceptibility to AIH-1 are DRB1*0301 and DRB1*0401,hich encode the HLA DR3 and DR4 antigens, respectively.18
he first genome wide association study (GWAS) in AIH per-ormed in Dutch AIH-1 patients and replicated in a cohort of
German patients confirmed the HLA association, DRB1*0301and DRB1*0401 being primary and secondary susceptibilitygenotypes, respectively.19
Susceptibility to AIH-2 is conferred by the possessionof HLA DR7 (DRB1*0701) and DR3 (DRB1*0301),20 and thosepatients who are positive for DRB1*0701 have a more aggres-sive form of the disease with worse overall prognosis.21
A number of genes outside the MHC have also been linkedto AIH susceptibility. For example, a substitution from A(adenine) to G (guanine) in exon 1 of the CTLA-4 geneconfers susceptibility to AIH-1 in Caucasians from NorthAmerica.22 Additionally, a polymorphism at position 308 inthe tumour necrosis factor � (TNFA) gene promoter is par-ticularly frequent in patients with AIH-1 from Europe andNorth America, and is associated with a poorer responseto steroids.23 A FAS gene promoter polymorphism at posi-tion 670 also enhances susceptibility to AIH and influencesprogression to a more aggressive form characterised by theearly development of cirrhosis.24 Polymorphisms in the vita-min D receptor can also be predisposing factors to thedevelopment of autoimmune liver disease.25 The first AIHGWAS reported that AIH-1 is associated with variants ofCARD10 and SH2B3 genes.19
A form of AIH resembling AIH-2 has been described insome 20% of patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), an autosomalrecessive condition due to homozygous mutations in theAIRE1 gene and characterised by a variety of organ-specificautoimmune diseases. Additionally, AIRE1 mutations havebeen reported in three children with severe AIH-2 withextra-hepatic autoimmune manifestations, as well as in fourchildren with AIH-1 with a family history of autoimmunedisease.26
Potential triggers
In patients with increased genetic susceptibility, a poten-tial trigger for AIH development is an immune response toexogenous pathogens that cross-reacts with structurally sim-ilar liver autoantigens, a phenomenon known as molecularmimicry. The strongest support for this model is in the con-text of viral hepatitis, where autoimmunity is a commonfeature during chronic infection. Indeed, 50% of patientswith chronic hepatitis B or C (HCV) eventually developautoantibody seropositivity.27,28 In chronic HCV, some 10%of patients are anti-LKM-1 positive, the autoantibody titrecorrelating with disease severity and being associated withadverse reactions to interferon treatment.29 The molecu-lar target of anti-LKM-1 is cytochrome P4502D6 (CYP2D6)and within anti-LKM-1 positive chronic HCV patients, reac-tivity against the key autoantigenic epitope CYP2D6193---212
can be seen in 50% of cases. There is direct evidence ofcross-reactivity between anti-LKM-1 and antibodies directedagainst homologous regions of HCV (NS5B HCV2985---2990) andcytomegalovirus (exon CMV130---135).30 There is also sequencehomology between CYP2D6254---271 and amino acids present inthe E1 HCV and the IE1 75 of the Herpes Simplex Virus 1
Please cite this article in press as: Liberal R, et al. Autoimmu2016. http://dx.doi.org/10.1016/j.rce.2016.04.003
(HSV-1). As anti-LKM-1 antibodies cross-react with homolo-gous regions of CYP2D6, HCV, CMV and HSV, a ‘‘multi-hit’’mechanism for the generation of autoantibody seropositivityand possibly for the development AIH-2 can be envisaged.
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n this model, on a background of genetic susceptibility,equential exposure to common viral pathogens favours theevelopment of cross-reactive T cells. It is therefore con-eivable that as yet unidentified single or repeated viralnfections could contribute to the initiation of the autoim-une attack in AIH.9 One case-report describes a 10-year-
ld girl who acquired HCV infection following liver transplantor end-stage liver disease caused by �1-anti-trypsin defi-iency. Two weeks after HCV infection IgM anti-LKM-1ntibodies appeared, followed by IgG anti-LKM-1 antibodies.his finding is suggestive of HCV as a trigger of a primary anti-KM-1/anti-CYP2D6 autoimmune response.31 Interestingly,0 years later, the patient developed florid AIH type 2, whichesponded satisfactorily to immunosuppressive treatment;y this time there was no trace of the previous HCV infectionunpublished data). Moreover, in a recent report, up to 8.7%f patients with autoimmune disease, including cryoglob-linaemia, Hashimoto thyroiditis and inflammatory bowelisease (IBD),32 had serum HCV antibody-positivity, linkingCV infection with a breakdown of immune tolerance.
The antibiotics nitrofurantoin and minocycline,33 as wells the statins and the anti-TNF agents adalimumab andnfliximab have been reported as non-viral environmentalriggers of AIH. However, because drug-induced liver injuryith features of AIH does not usually require long-term
mmunosuppressive treatment, these triggers should be con-idered independently.33
Though the evidence for molecular mimicry is convincing, universal trigger has not yet been discovered. Moreover,ther non-mutually exclusive mechanisms that may con-ribute to the initiation and perpetuation of AIH, such aspitope spreading or exposure to previously hidden autoanti-ens during hepatocellular injury, should also be explored.
ffector immune mechanisms
he dense infiltrate of lymphocytes, plasma cells, andacrophages characteristic of the histological picture ofIH suggests that an autoaggressive cellular immune attack
s the basis of this condition. Over the past four decades,ntense investigations have begun to uncover the mecha-isms by which this inflammatory infiltration mediates liveramage.
The predominant population within the cellular infiltrates composed of �/� T cells.34 Amongst these cells, the major-ty are CD4pos T helper (Th) cells, with a sizable minorityf cytotoxic CD8pos T-cells. Immunohistochemically, lympho-ytes of a non-T-cell lineage are seen relatively rarely, andnclude natural killer (NK)-cells, macrophages, B-cells andlasma cells.34
Regardless of the nature of the initial trigger, the immuneesponse in AIH is believed to stem from the recognitiony naïve CD4pos lymphocytes of a self-antigenic peptideontained within an HLA class II molecule on an antigenresenting cell (APC). In the presence of the appropri-te co-stimulatory signals --- provided by the interaction ofD28 expressed by the naïve CD4pos T-cell and CD80/CD86
ne hepatitis: From mechanisms to therapy. Rev Clin Esp.
xpressed by the APC --- the naïve cell differentiates into anffector cell subtype depending on the cytokines present inhe microenvironment and the nature of the antigen. Theiver is home to several specialised APC populations, and
Figure 1 Mechanisms of liver damage. Liver damage is initiated by the presentation of a self-antigenic peptide within a majorhistocompatibility molecule (MHC) by professional antigen presenting cells (APCs). The presence of appropriate costimulationalongside exposure to various cytokines drives the differentiation of uncommitted CD4 helper T-cells (Th0). IL-6 and IL-1� lead todifferentiation into pathogenic Th17 cells that secrete the proinflammatory cytokine IL-17. Th17 cells promote hepatocyte secretionof IL-6, which in turn further enhances Th17 development. Exposure to IL-12 leads to the differentiation of Th1 cells secreting IFN-�, which induces monocyte (M�) differentiation, activates cytotoxic CD8 T-cells and promotes NK cell killing. IFN-� also increasesMHC class I and induces class II expression by hepatocytes, further exacerbating inflammation. Exposure to IL-4 leads to Th2d e B co dy-m
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ifferentiation. Th2 cells secrete IL-13, IL-4 and IL-10 that enablf autoantibodies. Autoantibodies are in turn involved in antibo
ntigen presentation can occur in situ without the need ofrafficking to the regional lymphoid tissue.35
The effector T-cell subsets are largely defined by theytokines they produce; Th1-cells produce IL-2 as well ashe main mediator of the tissue damage in AIH, IFN-�.FN-� stimulates CD8pos T-cells, enhances the expressionf HLA class I molecules by hepatocytes, while inducinghe aberrant expression of HLA class II molecules and acti-ating monocytes/macrophages, which in turn release IL-1nd tumour necrosis factor alpha (TNF-�). On the otherand, Th2-cells produce IL-4, IL-10 and IL-13, cytokines thatnduce the maturation of B-cells into plasma cells, withonsequent production of autoantibodies. Autoantibodieshemselves can contribute to liver damage by triggeringntibody-mediated cellular cytotoxicity and complementctivation. Th17-cells produce IL-17, IL-22, and TNF-�,9
hile inducing hepatocytes to secrete IL-6, which furthernhances Th17 activation. Although Th17-cells have beenhown to be elevated in the circulation and liver of AIHatients, their precise contribution to the pathogenesis ofIH is unknown and currently under investigation. Mecha-isms leading to and/or perpetuating the autoimmune liverttack in AIH are depicted in Fig. 1.
egulatory immune mechanisms
Please cite this article in press as: Liberal R, et al. Autoimmu2016. http://dx.doi.org/10.1016/j.rce.2016.04.003
he development of autoimmune diseases is favoured by thereakdown of self-tolerance mechanisms that, in health,revent the majority of autoreactive T cell clones fromntering the periphery. As circulating autoreactive T cells
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ell maturation into plasma cells with the consequent productionediated cellular cytotoxicity and complement activation.
re, however, present in health, there are both intrinsic andxtrinsic peripheral tolerance mechanisms to limit autoim-une tissue damage. Key to this is the dominant form of
mmune suppression exerted by professional regulatory Tells (Tregs).36
Several studies have reported that regulatory T-cells areumerically defective in patients with AIH. A lower circu-ating frequency of CD4posCD25pos,37,38 CD4posCD25high39 orD4posCD25highCD127neg40 Tregs has been reported in AIHatients compared to healthy subjects, and this defects associated with lower FOXP3 expression.39,41 Moreover,uring immunosuppressive therapy, the circulating Treg fre-uency has been shown to increase to some extent, althoughot reaching the levels seen in health.37,39
When co-cultured with CD4posCD25neg cells, magneti-ally isolated CD4posCD25pos Tregs from AIH patients havempaired ability to generate an anti-inflammatory cytokineilieu rich in TGF-�, suggesting that they are ineffectiveromoters of linked-suppression.41 Magnetically isolatedD4posCD25pos Tregs from AIH patients have impairedbility to suppress the proliferation of CD8pos38 and ofD4posCD25neg39,42 cell populations compared to normal con-rols. CD4posCD25pos Tregs from AIH patients are also ineffec-ive suppressors of IFN-� production by CD8 T cells.38 Addi-ionally, Tregs from AIH patients, but not healthy subjects,ffectively enhance the activation of pro-inflammatoryonocytes, by elevating the level of spontaneous migration
ne hepatitis: From mechanisms to therapy. Rev Clin Esp.
nd by increasing the production of TNF-� and the expres-ion of TLR-4.43 Magnetically isolated CD4posCD25posCD127neg
regs from AIH patients are also less able to suppressD4posCD25neg cell proliferation in AIH compared to health,42
although this defect has not been observed in flow cytomet-rically isolated CD4posCD25highCD127neg cells.44 Tregs fromAIH patients express lower levels of galectin-9 compared tohealth, and this defect is mirrored by reduced expressionof the galectin-9 ligand, T-cell immunoglobulin and mucindomain-3 (TIM-3), by CD4posCD25neg cells, suggesting thatAIH may also be associated with effector cell resistanceto Treg control.42 We have recently observed that in AIHthere is a lower frequency of CD39pos Tregs, these cells fail-ing to hydrolyse adequately pro-inflammatory nucleotidesand to suppress the production of IL-17 by effector CD4T-cells. CD39pos Tregs from AIH patients are also unstableupon pro-inflammatory challenge,45 suggesting that defec-tive immuno-regulation in AIH might result not only fromreduced Treg number and function but also from increasedconversion of Tregs into effector cells.
Clinical presentation
AIH can present with diverse clinical manifestations.46 Thereare basically three patterns of disease presentation: anacute onset, characterised by non-specific symptoms suchas malaise, nausea/vomiting, anorexia and abdominal pain,followed by jaundice, dark urine and pale stools; an insidiousonset, with an illness characterised by progressive fatigue,relapsing jaundice, headache, anorexia, amenorrhoea andweight loss; and finally a presentation with complicationsof portal hypertension.17 The mode of presentation of AIHis therefore variable, and the disease should be suspectedand excluded in all patients complaining of symptoms andsigns of prolonged or severe liver disease. Some patients,however, are completely asymptomatic and are diagnosedafter incidental discovery of abnormal liver function tests.
Histological evidence of cirrhosis is described in at least30% of patients, regardless of the mode of presentation, sug-gesting that subclinical disease has been present for sometime. Indeed, advanced fibrosis or cirrhosis can often befound in patients presenting acutely.10
AIH can develop occasionally during pregnancy.47 Post-partum development of AIH and exacerbation of existingdisease in patients whose condition improved during preg-nancy has also been described.48 Approximately 40% of AIHpatients have a family history of autoimmune disease andat least 20% have concomitant autoimmune diseases or willdevelop them during follow-up.49
The complications associated with AIH mirror those foundin other progressive liver diseases. Thus, chronic hepatitiscan progress to cirrhosis and to hepatocellular carcinoma(HCC) despite the use of immunosuppressive therapy. HCCis relatively rare in AIH.10 Yeoman et al.50 reported 15 casesof HCC, all with underlying cirrhosis, among 243 patientswith AIH who were followed up for 16 years. Wong et al.reported 6 cases of HCC, also all with underlying cirrhosis,among 322 patients followed up for 10 years.51 Surveillancefor HCC is therefore warranted.4
Diagnosis
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The diagnosis of AIH is based on the presence of elevatedserum transaminase and IgG levels, autoantibody seroposi-tivity and histological evidence of interface hepatitis. This
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enet has been factored into the IAIHG diagnostic criteriaor AIH,13,14 which were originally developed for use in theesearch setting. This scoring system incorporates a num-er of positive and negative scores (Table 1), enabling theesearcher to grade clinical, laboratory and histological fea-ures of AIH. The score has also proved useful in the clinicalontext when assessing patients with few or atypical fea-ures of the disease. The distinction between a definitend probable diagnosis of AIH predominantly relates to thextent of the increase in serum gamma-globulin/IgG levelsr autoantibody titre, as well as exposure to alcohol, hepa-otoxic medication or infection. Laboratory and histologicaleatures associated with cholestasis carry a negative score.n the rare instances where conventional autoantibodiesre not detected, the presence of anti-asialoglycoproteineceptor (anti-ASGPR), anti-soluble liver antigen/liver pan-reas (anti-SLA/LP) or atypical perinuclear anti-neutrophilytoplasmic antibodies (atypical pANCA, currently bettereferred to as pANNA) weigh towards a probable diagno-is of AIH. The scoring system also incorporates responseo corticosteroids, with a definite diagnosis before steroidreatment requiring a score higher than 15, and a defi-ite diagnosis after treatment institution requiring a scorereater than 17.14
It is important to note that because healthy children areery rarely autoantibody positive, in the paediatric setting,ower autoantibody titres contribute to the diagnoses of AIH.hese titres are 1:20 for ANA and SMA and 1:10 for anti-LKM-.2,12
A simplified scoring system (Table 2), for use in clin-cal practice, has recently been proposed by the IAIHG.his system is based on only four criteria: positivity forutoantibodies, elevated IgG levels, histological evidencef interface hepatitis and the exclusion of viral hepatitis.52
either scoring system is immediately applicable to theiagnosis of the juvenile form of AIH.
utoantibodies
utoantibody seropositivity is a key criterion for the diag-osis and classification of AIH. The IAIHG includes positivityor ANA, SMA, anti-LKM-1, anti-LC-1, anti-SLA/LP and ANCAsutoantibodies in their original and revised diagnostic sco-ing systems.13,14
nti-nuclear antibodies
he target antigens of ANA in AIH are heterogeneous andncompletely defined. Though ANA have been shown toeact with single- and double-stranded deoxyribonucleiccid (DNA), small nuclear ribonucleoproteins (sn-RNPs),entromeres, histones, chromatin and cyclin A, all theseeactivities can be negative despite positivity of ANAy immunofluorescence (IFL). A better definition of theNA target antigens will be followed by the develop-ent of new techniques using recombinant nuclear antigens
nd immunoassays.3 In terms of IFL, ANA gives a readily
ne hepatitis: From mechanisms to therapy. Rev Clin Esp.
etectable nuclear staining of kidney, stomach and liverections. In AIH, a homogenous pattern of staining is com-only observed, particularly in the liver, with coarsely ornely speckled patterns visualised less frequently.2 A clearer
Including granulomatous cholangitis, concentric periductal fibrb Any other prominent feature suggesting a different aetiology.
efinition of the nuclear pattern should be sought usinguman epithelial type 2 (HEp2) cells, which are charac-erised by prominent nuclei. These, however, should not besed for screening purposes due to a high positivity raten healthy subjects. A clinically relevant titre of ANA inIH is considered 1/40 in adults and 1/20 in children, inhom titres correlate with disease activity. ANA can also be
dentified in up to 52% of patients with PBC. However, inontrast to AIH, in which no disease-specific ANA has been
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eported, the PBC-specific ANA showing multiple nuclear dotr rim-like membranous patterns are highly diagnostic forhis condition. They are recognised by IFL when HEp-2 oreLa cells are used as substrate. Of note, ANA are present in
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ductopenia, marginal bile duct proliferation and cholangiolitis.
ther autoimmune disorders, such as systemic lupus erythe-atosus, Sjögren syndrome and systemic sclerosis, as well
s non-autoimmune conditions, like viral hepatitis, drug-nduced hepatitis, and alcoholic and non-alcoholic fatty liverisease.53
nti-smooth muscle antibodies
ne hepatitis: From mechanisms to therapy. Rev Clin Esp.
he first targets of AIH-specific SMA to be recognised-- following preincubation of serum with thrombostheninplatelet actomyosin) or purified thrombosthenin-A (thectin fraction of thrombosthenin) --- were constituents of
Table 2 Simplified criteria for the diagnosis of autoimmunehepatitis.
Variable Cut-off Points
ANA or SMA ≥1:40 1ANA or SMA ≥1:80 2a
Or anti-LKM-1 ≥1:40Or SLA PositiveIgG >Upper limit of normal 1
>1.10 times upper limitof normal
2
Liver histology Compatible with AIH 1Typical of AIH 2
Absence of viralhepatitis
Yes 2
Adapted from Hennes EM, Zeniya M et al. Hepatology2008;48:169---176.Score ≥6: probable AIH; ≥7: definite AIH.ANA, anti-nuclear antibody; SMA, anti-smooth muscle antibody;anti-LKM-1, anti-liver kidney microsomal antibody type 1; SLA,soluble liver antigen; IgG, immunoglobulin G; AIH, autoimmunehepatitis.
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a Addition of points achieved for all autoantibodies cannotexceed a maximum of 2 points.
actin. Later, SMA were also shown to be directed againstother components of the cytoskeleton such as tubulin,vimentin, desmin, and skeletin.54 SMA IFL patterns can bevisualised on kidney, stomach and liver sections, where theystain the artery walls. In the stomach substrate they alsobind the muscularis mucosa and the lamina propria. In thekidney, the SMA typical of AIH stains the smooth muscleof the vessels, glomeruli and tubules (VGT pattern). TheVG and VGT IFL patterns are much more specific for AIHthan the isolated V pattern.2 The AIH-1-specific target ofSMA responsible for the VGT pattern remains elusive. How-ever, when vinblastine-arrested cultured fibroblasts wereused as a substrate, AIH-1-specific SMA VGT-positive serapredominantly stained the microfilaments. In contrast, non-AIH-1 SMA V-positive sera reacted with non-actin-containingintermediate filaments. Several studies point to actin in itsfilamentous form as the target of the SMA giving the VGTpattern. However, while this pattern is highly specific forAIH-1, some 20% of SMA-positive AIH patients do not haveit. Moreover, when molecular assays using purified F-actinare employed, some AIH VGT positive cases are negative,while anti-F-actin positivity is reported in diseases distinctfrom AIH-1.2,54 SMA titres by IFL are usually equal or above1/80 in AIH, although very young patients may have titres aslow as 1/20. SMA giving the V pattern have been reportedin advanced liver disease of other aetiologies, infectiousdiseases and rheumatic disorders; hence, like ANA, theseantibodies are not specific for AIH.
Anti-liver-kidney-microsomal type 1 antibodies
The molecular target of anti-LKM-1, the hallmark of AIH-
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2, is CYP2D6. This autoantibody stains the hepatocellularcytoplasm and the P3 portion of the renal tubules. Someconfusion can occur between the IFL patterns of anti-LKM-1 and AMA because both autoantibodies stain the liver as
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ell as the kidney. However, AMA stain the liver moreaintly than anti-LKM-1, and mark the renal tubules moreiffusely, while accentuating the mitochondrial-reach dis-al tubules. Importantly, AMA stain gastric parietal cellshile anti-LKM-1 do not.2,54 Since the molecular targetsf anti-LKM-1 --- CYP2D6 --- and of AMA --- enzymes ofhe 2-oxo-acid dehydrogenase complexes --- are known,mmune-assays based on the use of recombinant or puri-ed antigens have been developed. Commercially availablenzyme-linked immunosorbent assays (ELISAs) accuratelyetect anti-LKM-1, at least in the context of AIH-2, andetect AMA reasonably accurately. These assays can there-ore be utilised when there is doubt about IFL patterns.2
clinically relevant anti-LKM-1 titre is considered equal orbove 1/40 in adults and 1/10 in patients under 18 yearsf age; the titre of this autoantibody is associated with dis-ase activity.2 Interestingly, as mentioned above, anti-LKM-1re also detected in some 5---10% of patients with chronicepatitis C virus infection.
nti-liver cytosol type 1 antibodies
nti-LC-1 react with the folate-metabolising enzymeormiminotransferase cyclodeaminase (FTCD), which isound at high levels within the liver. This autoantibodytains the liver cell cytoplasm with relative sparing of theentrilobular area. Importantly, anti-LC-1 frequently occursogether with anti-LKM-1, which obscure the anti-LC-1 stain-ng. Thus, in the presence of anti-LKM-1, anti-LC-1 can beetected by the use of liver cytosol in double-dimensionmmunodiffusion or counterimmunoelectrophoresis, withhe use of a positive reference serum, or by ELISA detec-ing reactivity to its target FTCD.2 This autoantibody wasriginally described, alone or in combination with anti-KM-1, to define a clinical entity indistinguishable fromIH-2. Although anti-LC-1 was subsequently detected also
n patients positive for serological markers associated withIH-1, and in patients with chronic HCV infection, anti-LC-
in isolation scores positively towards a diagnosis of AIH-2,llowing prompt initiation of treatment.14 The presence anditre of anti-LC-1 antibodies correlate with disease activ-ty, and represent a potentially useful marker of residualepatocellular inflammation in AIH.54
nti-SLA and anti-LP were originally believed to be dis-inct antibodies but they were subsequently shown to bindhe same target, a UGA tRNA suppressor-associated anti-enic protein (tRNP(ser)sec), more precisely O-Phosphoseryl-RNA:selenocysteinyl-tRNA synthase (SepSecS).55 They can,herefore, be considered one and the same. Since anti-LA/LP cannot be detected by IFL, this antibody is detectedy radioimmunoassay and ELISA.2 Anti-SLA/LP have beeneported in the absence of seropositivity for conventionalutoantibodies, suggesting the existence of a third groupf AIH patients. However, these early reports used a par-
ne hepatitis: From mechanisms to therapy. Rev Clin Esp.
icularly high cut-off point for conventional autoantibodyetection --- higher than those currently used for the diag-osis of AIH --- therefore the nosological entity of AIH-3 hasot been accepted by the IAIHG. Though anti-SLA have been
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reported in occasional HCV infected anti-LKM-1 positivepatients, their presence is highly specific for the diagnosisof AIH, and its detection at the time of diagnosis identifiespatients with a more severe disease and a worse prognosis.21
Anti-neutrophil cytoplasmic antibodies
ANCAs react to constituents of the cytoplasm of neutrophilsto give a perinuclear (pANCA) or cytoplasmic (cANCA)IFL pattern. The predominant target of c-ANCA is pro-teinase 3 and this autoantibody is frequently detected inWegener granulomatosis. On the other hand, p-ANCA bindsmyeloperoxidase and is most commonly found in micro-scopic polyangiitis. Similar to what observed in PSC and IBD,pANCAs are frequently detected in AIH-1, although the IFLpattern is somewhat atypical. Staining is associated withperipheral nuclear membrane components, hence the nameof peripheral anti-nuclear neutrophil antibody (pANNA). Theproposed target of pANNA is a 50 kDa neutrophil-specificnuclear protein belonging to the nuclear pore complex,potentially the tubulin � chain 5.54 Positivity for pANNA isvery rare in AIH-2. In AIH-1, however, its detection can aidin the diagnosis, particularly when other autoantibodies areabsent.14
Histology
Since transaminases and IgG levels do not reflect the extentof histological inflammatory activity, or the presence orabsence of cirrhosis, liver biopsy is mandatory not only toconfirm the diagnosis but also to evaluate the severity ofliver damage.
Hepatitis at the portal-parenchymal interface, known asinterface hepatitis (Fig. 2), is typical, but is not exclu-sively observed in AIH.56 This picture is characterised by alymphoplasmacytic infiltrate crossing the limiting plate andinvading the liver parenchyma. Lymphocytes, plasma cells
Figure 2 Histology of autoimmune hepatitis. The portal andperiportal inflammatory infiltrate characteristic of autoimmunehepatitis is composed of lymphocytes, monocytes/macrophagesand plasma cells (interface hepatitis, arrows). Haematoxilin andeosin staining (picture kindly provided by Dr. Alberto Quaglia,Institute of Liver Studies, King’s College Hospital).
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dtcwi
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nd histiocytes surround individual dying hepatocytes athe portal-parenchymal interface and in the lobule. Thoughlasma cells are usually abundant at the interface andhroughout the lobule, their presence in low number doesot exclude the diagnosis of AIH.17 Other lesions that maye present include hepatocyte swelling and pycnotic necro-is. Fibrosis is present in all but the mildest forms of theisease. In contrast to patients with an insidious course,hose presenting with acute liver failure show tissue damageredominantly in the centrilobular area.57 However, manyatients presenting with fulminant hepatic failure tend toave massive necrosis and multilobular collapse; impor-antly, they have less fibrosis than those presenting with aore insidious course.10
Despite the fact that sampling variation may occur ineedle biopsy specimens, especially in the presence ofirrhosis, the severity of the histological appearance issually of prognostic value. However, even patients with cir-hosis at presentation respond well to immunosuppressivereatment.58
reatment
tandard treatment
n the early 70s, three randomised clinical trials in adultsith AIH provided the basis for the current immunosup-ressive regimens, collectively suggesting that treatmentith prednisolone improves liver function tests, ameliorates
ymptoms and prolongs survival.59---61 Although azathioprineas not able to induce remission when used on its own, it didllow the maintenance of remission in association with a sig-ificantly reduced dose of steroids.62 Initial treatment withrednisone (or prednisolone), with or without azathioprine,hould be instituted as soon as the diagnosis is made, andot delayed for 6 months as suggested in the early studies.63
The initial approach to therapy depends upon the his-ologic findings.10 Transaminase and IgG levels do notlways correlate with histological damage and consequentlyrovide limited help in respect to treatment initiation.rompt treatment is indicated in patients with aggressiveisease, usually children or young adults, and in any patientith evidence of interface hepatitis, with or without fibrosisr cirrhosis.4 In older patients with isolated portal inflam-ation (without interface hepatitis), institution of therapy
s guided by AST and IgG levels, and/or by the presencef symptoms.46 The therapeutic approach in patients with
milder form of the disease --- who may be asymptomaticr pauci-symptomatic, and are detected incidentally afteriscovery of abnormal liver function tests --- is less clear.64
n these cases, the benefit of therapy is undefined anday be so low that the risk of corticosteroid side effectsight outweigh its possible benefits; this is particularly rel-
vant when starting therapy in post-menopausal women andlderly patients.65
Although some patients may remain in remission afterrug treatment is withdrawn, most require long-term main-
ne hepatitis: From mechanisms to therapy. Rev Clin Esp.
enance therapy. Despite the absence of firm guidelines, it isautious not to attempt withdrawal of immunosuppressionithin 2 years of diagnosis.4 During withdrawal attempts,
t is essential to closely monitor the liver function tests, as
relapse may be severe and even fatal. Patients who have suc-cessfully stopped immunosuppression should undergo long-term follow-up, as relapse can occur even 10 years later.64
Alternative treatments
In the group of patients that show failure to standardtherapy and intolerance or low compliance to standardimmunosuppression, alternative immunosuppressive treat-ments have been proposed.66 Decisions regarding the useof such regimens have to be based, however, on the scarcedata available, mainly on the basis of small series or casereports.
Mycophenolate mofetil (MMF) is a purine antagonist thatselectively inhibits proliferation of activated lymphocytes.67
It has been reported to be effective in AIH patients intol-erant to azathioprine.64 Therefore, in patients for whomstandard immunosuppression fails to induce stable remis-sion, or who are intolerant to azathioprine, MMF, togetherwith prednisolone, is currently the treatment of choice.4
Calcineurin inhibitors, cyclosporine and tacrolimus, havebeen used as a rescue treatment for difficult-to-treat casesof AIH. As no large study in this subgroup of patients hasbeen published, they should be used with caution.64
Anti-TNF-� agents, such as infliximab, are commonlyused to treat immune-mediated diseases such as rheumatoidarthritis, psoriasis and IBD. There is anecdotal evidence thatinfliximab is efficacious in the management of difficult-to-treat cases of AIH.68 In the largest published retrospectiveseries, treatment with infliximab led to a decrease intransaminase and IgG levels in 11 difficult-to-treat adultpatients with AIH, but infectious complications occurredin seven of them.68 Moreover, and worryingly, infliximabtherapy for other diseases has been associated with theinduction of severe de novo AIH.69 Anecdotal evidence alsosuggests some benefit with the use of the anti B-cell mono-clonal antibody rituximab in difficult-to-treat patients.70
However, the occurrence of severe infections is an importantrisk-factor associated with these biological treatments.
Budesonide is a corticosteroid with very high affinityfor the glucocorticoid receptor and high first pass livermetabolism; hence, it is presently receiving considerableattention as an alternative to prednisone or prednisoloneas primary treatment of AIH. Although initial reports weresomewhat contradictory, a recent large European studyfound that a combination of budesonide and azathioprinecould induce remission in 60% of non-cirrhotic patients,while medium-dose standard steroids and azathioprine couldonly induce remission in 39% of patients. The budesonidegroup had also fewer adverse effects.71 It should be noted,however, that this reported rate of remission is much lowerthan that seen in both adults and children (∼80%) whena higher starting dose of prednisone is used. Moreover,because budesonide cannot be used in cirrhotic patients--- representing at least a third of the AIH population --- itsclinical utility has limitations.72
Please cite this article in press as: Liberal R, et al. Autoimmu2016. http://dx.doi.org/10.1016/j.rce.2016.04.003
Liver transplantation
AIH accounts for 2---3% of paediatric and 4---6% of adult livertransplants (LT) performed in Europe and the United States.4
rTbd
PRESS9
T is indicated for AIH patients who present with acuteiver failure and do not respond to immunosuppressive treat-ent, or with end-stage chronic liver disease and for thoseith hepatocellular carcinoma that meet the transplantriteria.73,74 Although patients with a chronic presentationf AIH typically respond well to immunosuppressive treat-ent, approximately 10% will eventually require LT. Patientsho fail to reach remission after 4 years of therapy are
he most common candidates for LT.75 The indications forT for end-stage chronic AIH are similar to those for othernd-stage liver diseases.1,15
ovel treatment approaches
iven that loss of immunoregulation is central to theathogenesis of AIH, Treg cell therapy --- aimed at recon-tituting self-tolerance --- is a highly promising candidateor alternative and effective immune intervention in AIH.o date, this approach has been hindered by the lim-ted ability of Tregs to expand and by their propensity topoptosis. However, because corticosteroid therapy can par-ially restore the potency of the Treg population, Tregsn AIH do have the potential to expand and regain theirunction.37,38 Using a polyclonal T cell stimulation strat-gy (that engages the T cell receptor via CD3 and theo-stimulatory molecule CD28, while providing exogenousL-2, a key cytokine for Treg survival and growth), Tregs cane expanded from circulating CD4posCD25pos Tregs, and alsoenerated de novo from non-regulatory CD4posCD25neg T cellsn both healthy subjects and patients with AIH.76 Interest-ngly, expanded Tregs express higher levels of FOXP3 andre more effective suppressors compared to freshly isolatedregs.76
Although FOXP3 is the most specific marker of humanregs, its intracellular location limits its use in the laboratoryetting. In addition to the lack of specific cell-surface mark-rs for Tregs, the human CD4pos CD25high population contains
proportion of activated effector T cells. Furthermore,regs and effector Th17 cells share a common progenitor,hough their developmental pathways differ. Since de novoeneration of Tregs relies on strong T cell receptor (TCR)ignalling, the risk of concomitant expansion of effectorh17 cells needs to be addressed when considering Tregherapy for AIH.9 The physical removal of IL17pos cells, or these of small interfering RNAs specific for the Th17-associatedranscription factor RORC, leads to elevated FOXP3 expres-ion and increased suppressive function by expanded Tregsrom AIH patients.77
The potential for successful Treg therapy is particu-arly strong in AIH-2, given that the antigenic regionsCYP2D6217---260 and CYP2D6305---348), targeted by B, CD4 andD8 T cells, are well characterised.78 Several lines of evi-ence demonstrate that autoantigen-specific Tregs suppressore efficiently than their non-antigen-specific counter-arts. In this regard, antigen-specific Tregs generated fromIH-2 patients are able to suppress CD4 and CD8 T cell
ne hepatitis: From mechanisms to therapy. Rev Clin Esp.
esponses more potently than polyclonally expanded T-regs.he most efficient suppression of autoreactive T cells haseen achieved by Treg co-culture with semi-mature den-ritic cells loaded with the CYP2D6 peptides.79
. Liberal, D. Vergani and G. Mieli-Vergani are supported byrants from the Rosetree Foundation, UK, and PSC Partnerseeking a Cure, USA.
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of CD25-cells into stable T regulatory cells in patients withautoimmune hepatitis. Gastroenterology. 2012;142:1526---35.
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