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Primary biliary cholangitis: A tale of epigenetically-induced secretory failure?

  • Pedro M. Rodrigues
    Affiliations
    Department of Liver and Gastrointestinal Diseases, Biodonostia Health Research Institute – Donostia University Hospital, University of the Basque Country (UPV/EHU), San Sebastian, Spain
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  • Maria J. Perugorria
    Affiliations
    Department of Liver and Gastrointestinal Diseases, Biodonostia Health Research Institute – Donostia University Hospital, University of the Basque Country (UPV/EHU), San Sebastian, Spain

    National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd, “Instituto de Salud Carlos III”), Spain

    Ikerbasque, Basque Foundation for Science, Bilbao, Spain
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  • Alvaro Santos-Laso
    Affiliations
    Department of Liver and Gastrointestinal Diseases, Biodonostia Health Research Institute – Donostia University Hospital, University of the Basque Country (UPV/EHU), San Sebastian, Spain
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  • Luis Bujanda
    Affiliations
    Department of Liver and Gastrointestinal Diseases, Biodonostia Health Research Institute – Donostia University Hospital, University of the Basque Country (UPV/EHU), San Sebastian, Spain

    National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd, “Instituto de Salud Carlos III”), Spain
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  • Ulrich Beuers
    Affiliations
    Tytgat Institute for Liver and Intestinal Research and Department of Gastroenterology & Hepatology, Amsterdam Gastroenterology and Metabolism, AMC, Amsterdam, The Netherlands
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  • Jesus M. Banales
    Correspondence
    Corresponding author. Address: Department of Liver and Gastrointestinal Diseases, Biodonostia Health Research Institute – Donostia University Hospital, Paseo del Dr. Begiristain s/n, E-20014 San Sebastian, Spain; Tel.: +34 943006067. This author was the recipient of the EASL Young Investigators’ Award 2018.
    Affiliations
    Department of Liver and Gastrointestinal Diseases, Biodonostia Health Research Institute – Donostia University Hospital, University of the Basque Country (UPV/EHU), San Sebastian, Spain

    National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd, “Instituto de Salud Carlos III”), Spain

    Ikerbasque, Basque Foundation for Science, Bilbao, Spain
    Search for articles by this author
Published:September 04, 2018DOI:https://doi.org/10.1016/j.jhep.2018.08.020

      Summary

      Primary biliary cholangitis (PBC) is a chronic cholestatic liver disease associated with autoimmune-related destruction of small to medium size intrahepatic bile ducts. The aetiology of PBC is unknown and its pathogenesis remains obscure. Both genetic variants and environmental factors have been linked to increased PBC susceptibility, with other alterations known to cooperate in disease pathobiology. Increasing evidence indicates the presence of epigenetic abnormalities in PBC, particularly alterations of cholangiocellular microRNAs (miRNAs or miRs). This review highlights and discusses the most relevant epigenetic alterations found in patients with PBC, focusing on the role of miR-506 in the promotion of cholestasis and immune activation.

      Keywords

      Primary biliary cholangitis – an integrative overview

      PBC is a chronic cholestatic liver disease associated with autoimmune phenomena that target the small and medium size bile ductules. The aetiology of PBC remains unknown and the pathogenesis obscure.
      Primary biliary cholangitis (PBC) is a chronic and slowly progressive liver disease associated with autoimmune events that are responsible for the selective destruction of small and medium size bile ductules, resulting in intrahepatic cholestasis and liver injury. As in other liver disorders, ductular proliferation occurs in PBC as a primordial response to cholestasis. However, the progressive disease course and the autoimmune phenomena subsequently lead to cholangiocyte death, peribiliary fibrosis, ductopenia, cirrhosis, and occasionally hepatocellular carcinoma (HCC), resulting in hepatic failure and premature death.
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      Aetiopathogenesis

      The aetiology of PBC remains obscure. Throughout the past decades, environmental factors have been widely linked to disease aetiopathogenesis and to the breakdown of immune tolerance in PBC. In this regard, infectious agents are hypothesized as potential disease triggers. Patients with PBC have a higher prevalence of urinary tract infections, mainly Escherichia coli-related ones.
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      Genetic, environmental and epigenetic factors are believed to cooperate and trigger PBC.

      Genetic susceptibility

      The prominence of genetic factors in PBC was highlighted by several studies, endorsing the idea that PBC is a partially heritable disorder. In the past decade, studies in twins have shown higher PBC frequency in monozygotic twins than in dizygotic ones (with a pairwise concordance rate of 0.63, representing one of the highest rates found in autoimmune diseases),
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      strongly suggesting a marked genetic susceptibility. Genome-wide association studies (GWAS) were able to identify dozens of predisposing single-nucleotide polymorphisms (SNPs). Among them, the strongest association is found within the human leukocyte antigen (HLA) class II loci.
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      A genome-wide association study identifies six novel risk loci for primary biliary cholangitis.
      Although these results point to a marked genetic susceptibility in PBC, cases of discordant monozygotic twins were found, which showed differences in gene expression profiles, copy number variants and DNA methylation patterns between the affected and unaffected twins.
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      Genome-wide analysis of DNA methylation, copy number variation, and gene expression in monozygotic twins discordant for primary biliary cirrhosis.
      Notably, although interleukin-12 (IL-12) was previously identified as a risk locus in GWAS studies and possibly related with PBC pathogenesis and evolution,
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      a recent proof-of-concept study reported that the administration of the anti-IL-12 monoclonal antibody ustekinumab to patients with PBC did not reach the proposed primary endpoint,
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      • Levy C.
      • Zou B.
      • et al.
      Ustekinumab for patients with primary biliary cholangitis who have an inadequate response to ursodeoxycholic acid: a proof-of-concept study.
      reinforcing the fact that PBC does not only have a genetic background, but also relies on other mechanisms. Conversely, frequent X monosomy was reported in female patients with PBC, particularly in B and T cells, which could partly explain the female preponderance in PBC.
      • Invernizzi P.
      • Miozzo M.
      • Battezzati P.M.
      • Bianchi I.
      • Grati F.R.
      • Simoni G.
      • et al.
      Frequency of monosomy X in women with primary biliary cirrhosis.
      The X chromosome loss happens in a preferential fashion, more frequently involving one parental chromosome.
      • Miozzo M.
      • Selmi C.
      • Gentilin B.
      • Grati F.R.
      • Sirchia S.
      • Oertelt S.
      • et al.
      Preferential X chromosome loss but random inactivation characterize primary biliary cirrhosis.
      Future genetic data analysis and X chromosome association studies
      • Gao F.
      • Chang D.
      • Biddanda A.
      • Ma L.
      • Guo Y.
      • Zhou Z.
      • et al.
      XWAS: a software toolset for genetic data analysis and association studies of the X chromosome.
      should be performed in PBC to validate these data and deepen our understanding of these alterations.

      Breakdown of PDC-E2 immune tolerance

      PBC is strongly related with the loss of PDC-E2 immune tolerance. Defective clearance of apoptotic bodies released from dying BECs has been proposed as a possible source of autoantigens and further autoimmunity. Thus, intact and antigenically reactive PDC-E2 was detected in the membrane surface of apoptotic blebs from cultured human BECs, exposing an apotope that was absent in other cell types.
      • Lleo A.
      • Selmi C.
      • Invernizzi P.
      • Podda M.
      • Coppel R.L.
      • Mackay I.R.
      • et al.
      Apotopes and the biliary specificity of primary biliary cirrhosis.
      These apotopes could stimulate the immune system and are accessible to AMAs, suggesting a potential explanation for the selective biliary autoimmunity in PBC. Further, apoptotic bodies released from BECs in combination with AMAs stimulate a burst of pro-inflammatory cytokines from PBC macrophages.
      • Lleo A.
      • Bowlus C.L.
      • Yang G.X.
      • Invernizzi P.
      • Podda M.
      • Van de Water J.
      • et al.
      Biliary apotopes and anti-mitochondrial antibodies activate innate immune responses in primary biliary cirrhosis.
      Apart from PDC-E2, other mitochondrial autoantigens, including the E2 subunit of the oxo-glutarate dehydrogenase complex and the E2 subunit of the branched chain 2-oxo acid dehydrogenase complex, were also presented in apoptotic bodies from BECs challenged with toxic bile acids.
      • Rong G.
      • Zhong R.
      • Lleo A.
      • Leung P.S.
      • Bowlus C.L.
      • Yang G.X.
      • et al.
      Epithelial cell specificity and apotope recognition by serum autoantibodies in primary biliary cirrhosis.
      Alterations in BEC apoptosis and phagocytosis of apoptotic cells constitute a plausible explanation for the tissue specificity found in PBC and for the breakdown of tolerance to mitochondrial antigens. Nonetheless, these mechanisms might be more relevant to the perpetuation of injury than to disease initiation. Several theories suggest that chemical compounds and/or infectious agents might be involved in the breakdown of tolerance, mostly through molecular mimicry and cross reactivity mechanisms.
      • Selmi C.
      • De Santis M.
      • Cavaciocchi F.
      • Gershwin M.E.
      Infectious agents and xenobiotics in the etiology of primary biliary cirrhosis.
      • Shimoda S.
      • Nakamura M.
      • Ishibashi H.
      • Kawano A.
      • Kamihira T.
      • Sakamoto N.
      • et al.
      Molecular mimicry of mitochondrial and nuclear autoantigens in primary biliary cirrhosis.

      The role of anion exchanger 2

      During the last two decades, defects in cholangiocyte bicarbonate secretion have been shown to be, at least partially, responsible for the PBC phenotype. In particular, anion exchanger 2 (AE2, also known as SCL4A2) has gained much attention in this field.

      AE2 – secretory functions and intracellular pH (pHi) regulation

      Cholangiocytes represent a small proportion of total liver cells (3–5%) but, importantly, they participate in the generation of the total bile flow in humans (up to 30%), through the alkalisation and fluidisation of the primary bile generated at the canaliculi of hepatocytes, resulting in a bicarbonate-rich fluid.
      • Tabibian J.H.
      • Masyuk A.I.
      • Masyuk T.V.
      • O'Hara S.P.
      • LaRusso N.F.
      Physiology of cholangiocytes.
      • Banales J.M.
      • Prieto J.
      • Medina J.F.
      Cholangiocyte anion exchange and biliary bicarbonate excretion.
      Several members of the electroneutral Na+-independent Cl/HCO3 AE family have already been described in human tissues, but only AE2 was found to be expressed in human cholangiocytes,
      • Medina J.F.
      Role of the anion exchanger 2 in the pathogenesis and treatment of primary biliary cirrhosis.
      being considered the main biliary HCO3 exporter.
      • Arenas F.
      • Hervias I.
      • Uriz M.
      • Joplin R.
      • Prieto J.
      • Medina J.F.
      Combination of ursodeoxycholic acid and glucocorticoids upregulates the AE2 alternate promoter in human liver cells.
      Immunohistochemistry experiments performed in human liver samples demonstrated that AE2 is expressed in cholangiocytes and mainly localised in the apical domain.
      • Martinez-Anso E.
      • Castillo J.E.
      • Diez J.
      • Medina J.F.
      • Prieto J.
      Immunohistochemical detection of chloride/bicarbonate anion exchangers in human liver.
      Experimental downregulation of AE2 in human and rat cholangiocytes demonstrated that AE2 is responsible for the Cl/HCO3 exchange activity and the resultant bicarbonate secretion in these cells,
      • Arenas F.
      • Hervias I.
      • Uriz M.
      • Joplin R.
      • Prieto J.
      • Medina J.F.
      Combination of ursodeoxycholic acid and glucocorticoids upregulates the AE2 alternate promoter in human liver cells.
      • Banales J.M.
      • Arenas F.
      • Rodriguez-Ortigosa C.M.
      • Saez E.
      • Uriarte I.
      • Doctor R.B.
      • et al.
      Bicarbonate-rich choleresis induced by secretin in normal rat is taurocholate-dependent and involves AE2 anion exchanger.
      • Uriarte I.
      • Banales J.M.
      • Saez E.
      • Arenas F.
      • Oude Elferink R.P.
      • Prieto J.
      • et al.
      Bicarbonate secretion of mouse cholangiocytes involves Na(+)-HCO(3)(−) cotransport in addition to Na(+)-independent Cl(−)/HCO(3)(−) exchange.
      whereas mouse cholangiocytes contain a Na+/HCO3 cotransporter, in addition to AE2, involved in bicarbonate secretion.
      • Uriarte I.
      • Banales J.M.
      • Saez E.
      • Arenas F.
      • Oude Elferink R.P.
      • Prieto J.
      • et al.
      Bicarbonate secretion of mouse cholangiocytes involves Na(+)-HCO(3)(−) cotransport in addition to Na(+)-independent Cl(−)/HCO(3)(−) exchange.
      Biliary HCO3 secretion generates a luminal alkaline “umbrella” that avoids the protonation of apolar hydrophobic bile salt monomers, which will no longer be able to invade cholangiocytes and induce cytotoxicity.
      • Beuers U.
      • Hohenester S.
      • de Buy Wenniger L.J.
      • Kremer A.E.
      • Jansen P.L.
      • Elferink R.P.
      The biliary HCO(3)(−) umbrella: a unifying hypothesis on pathogenetic and therapeutic aspects of fibrosing cholangiopathies.
      • Hohenester S.
      • Wenniger L.M.
      • Paulusma C.C.
      • van Vliet S.J.
      • Jefferson D.M.
      • Elferink R.P.
      • et al.
      A biliary HCO3− umbrella constitutes a protective mechanism against bile acid-induced injury in human cholangiocytes.
      The presence of an intact glycocalyx at the cholangiocyte apical membrane and the intracellular bicarbonate sensor soluble adenylyl cyclase along with functional AE2 are vital to maintain the biliary HCO3 umbrella and to protect cholangiocytes against toxic bile acid-induced apoptosis.
      • Hohenester S.
      • Wenniger L.M.
      • Paulusma C.C.
      • van Vliet S.J.
      • Jefferson D.M.
      • Elferink R.P.
      • et al.
      A biliary HCO3− umbrella constitutes a protective mechanism against bile acid-induced injury in human cholangiocytes.
      • Chen Y.
      • Cann M.J.
      • Litvin T.N.
      • Iourgenko V.
      • Sinclair M.L.
      • Levin L.R.
      • et al.
      Soluble adenylyl cyclase as an evolutionarily conserved bicarbonate sensor.
      • Mardones P.
      • Medina J.F.
      • Elferink R.P.
      Activation of cyclic AMP Signaling in Ae2-deficient mouse fibroblasts.
      • Chang J.C.
      • Go S.
      • de Waart D.R.
      • Munoz-Garrido P.
      • Beuers U.
      • Paulusma C.C.
      • et al.
      Soluble adenylyl cyclase regulates bile salt-induced apoptosis in human cholangiocytes.
      In this context, AE2 is also responsible for maintaining the intracellular pH (pHi) within a narrow physiological range in cholangiocytes.
      The Cl/HCO3 anion exchanger 2 (AE2) is responsible for the alkalinisation of the bile, the generation of the “biliary bicarbonate umbrella”, and the regulation of the intracellular pH in human cholangiocytes.

      AE2 and PBC – a relationship of secretory failure

      AE2 expression and activity are decreased in cholangiocytes from patients with PBC, resulting in impaired secretin-stimulated biliary bicarbonate secretion, which improve under chronic treatment with UDCA.
      In the early 90s, Prieto et al.
      • Prieto J.
      • Qian C.
      • Garcia N.
      • Diez J.
      • Medina J.F.
      Abnormal expression of anion exchanger genes in primary biliary cirrhosis.
      hypothesized that disturbances in biliary bicarbonate secretion occur in patients with PBC and are responsible for the characteristic cholestasis. This hypothesis was mainly based on the improved clinical course of patients treated with UDCA, a choleretic bile acid.
      • Corpechot C.
      • Carrat F.
      • Bahr A.
      • Chretien Y.
      • Poupon R.E.
      • Poupon R.
      The effect of ursodeoxycholic acid therapy on the natural course of primary biliary cirrhosis.
      Accordingly, the expression of AE2 in liver biopsies and peripheral blood mononuclear cells (PBMCs) was found to be significantly reduced in patients with PBC compared to healthy controls.
      • Prieto J.
      • Qian C.
      • Garcia N.
      • Diez J.
      • Medina J.F.
      Abnormal expression of anion exchanger genes in primary biliary cirrhosis.
      • Medina J.F.
      • Martinez A.
      • Vazquez J.J.
      • Prieto J.
      Decreased anion exchanger 2 immunoreactivity in the liver of patients with primary biliary cirrhosis.
      Additionally, cultured cholangiocytes from patients with PBC showed reduced AE2 activity compared to normal human cholangiocytes.
      • Melero S.
      • Spirli C.
      • Zsembery A.
      • Medina J.F.
      • Joplin R.E.
      • Duner E.
      • et al.
      Defective regulation of cholangiocyte Cl−/HCO3(−) and Na+/H+ exchanger activities in primary biliary cirrhosis.
      Moreover, positron emission tomography studies using labelled bicarbonate demonstrated that patients with PBC present a deficient secretin-stimulated biliary bicarbonate secretion, which improves in patients undergoing UDCA treatment.
      • Prieto J.
      • Garcia N.
      • Marti-Climent J.M.
      • Penuelas I.
      • Richter J.A.
      • Medina J.F.
      Assessment of biliary bicarbonate secretion in humans by positron emission tomography.
      All these data point to impaired biliary bicarbonate secretion in patients with PBC, which consequently promotes cholestasis, while altered pHi in cholangiocytes may lead to severe cell disturbances and cholangiocyte distress. Remarkably, Ae2a,b−/− mice spontaneously develop several hepatobiliary and immunological PBC-like features, including portal infiltration of CD4+ and CD8+ T lymphocytes and bile duct damage, increased oxidative stress in cholangiocytes, periductular hepatic fibrosis, elevated production of interferon-γ (IFN-γ) and IL-12, increased serum levels of IgM, IgG and hepatic ALP, and development of serum AMAs specific against PDC-E2.
      • Salas J.T.
      • Banales J.M.
      • Sarvide S.
      • Recalde S.
      • Ferrer A.
      • Uriarte I.
      • et al.
      Ae2a, b-deficient mice develop antimitochondrial antibodies and other features resembling primary biliary cirrhosis.
      The characteristic AE2 deficiency of PBC immunocytes
      • Prieto J.
      • Qian C.
      • Garcia N.
      • Diez J.
      • Medina J.F.
      Abnormal expression of anion exchanger genes in primary biliary cirrhosis.
      might also have an important role in the autoimmune phenomena. In fact, CD8+ T cells rely on AE2 to maintain the pHi under physiological levels, as Ae2-deficient mouse CD8+ T cells show an alkalinised pHi and signs of increased proliferation and activation.
      • Concepcion A.R.
      • Salas J.T.
      • Sarvide S.
      • Saez E.
      • Ferrer A.
      • Lopez M.
      • et al.
      Anion exchanger 2 is critical for CD8(+) T cells to maintain pHi homeostasis and modulate immune responses.
      Aged Ae2a,b−/− mice exhibit higher number of intrahepatic cytotoxic CD8+ T cells, which show repression of programmed death-1 expression, concomitantly with decreased apoptosis, consequently favouring chronic immune-mediated cholangitis.
      • Concepcion A.R.
      • Salas J.T.
      • Saez E.
      • Sarvide S.
      • Ferrer A.
      • Portu A.
      • et al.
      CD8+ T cells undergo activation and programmed death-1 repression in the liver of aged Ae2a, b−/− mice favoring autoimmune cholangitis.
      Moreover, AE2 downregulation is associated with disturbed autophagy, anomalous expression of PDC-E2 and cholangiocyte senescence,
      • Sasaki M.
      • Sato Y.
      • Nakanuma Y.
      An impaired biliary bicarbonate umbrella may be involved in dysregulated autophagy in primary biliary cholangitis.
      which are characteristic of PBC. Notably, the expression of AE2 is upregulated in human cholangiocytes exposed to UDCA together with glucocorticosteroids, leading to enhanced biliary bicarbonate secretion.
      • Arenas F.
      • Hervias I.
      • Uriz M.
      • Joplin R.
      • Prieto J.
      • Medina J.F.
      Combination of ursodeoxycholic acid and glucocorticoids upregulates the AE2 alternate promoter in human liver cells.
      . Additionally, stimulation of biliary phospholipid secretion by fibrates has been demonstrated, which might contribute to stabilization of the defective biliary bicarbonate umbrella in PBC by reducing bile salt monomer levels in bile.
      • Ghonem N.S.
      • Assis D.N.
      • Boyer J.L.
      Fibrates and cholestasis.
      Overall, these data support the theory that decreased AE2 expression and activity in PBC might lead firstly to impaired biliary bicarbonate secretion with consequent cholestasis and cholangiocyte injury, and secondly to the breakdown of immune tolerance and autoimmune responses against susceptible cholangiocytes, resulting in profound biliary damage and ductopenia. Of note, until now, no SNPs have been mapped to the AE2 locus, strongly suggesting that AE2 modulation in PBC occurs mainly through epigenetic modifications.

      Epigenetics and PBC

      Dynamic environmental changes contribute markedly to the phenotype of patients, mainly through epigenetic modifications. Consequently, all the dynamic changes in the surrounding environment might strongly modify the epigenome, altering gene expression. The study of epigenetic processes, including DNA methylation patterns, histone variants, non-coding RNAs and post-translational modifications is now of considerable interest in PBC and will be reviewed and discussed further.

      DNA methylation

      The addition of a methyl group to a cytosine residue, converting it to 5-methylcytosine, is usually performed by DNA methyl-transferases, occurring preferably at CpG islands within gene promoters or intragenic regions. While hypermethylated states found within gene promoters are always related to gene expression silencing, hypermethylated states in intragenic regions are usually associated with increased expression levels.
      • Aran D.
      • Toperoff G.
      • Rosenberg M.
      • Hellman A.
      Replication timing-related and gene body-specific methylation of active human genes.
      Alterations in DNA methylation have been described in PBC (Table 1). For instance, from a panel of 125 X chromosome genes, the mRNA expression of CLIC2, a Cl intracellular channel that modulates Ca2+ homeostasis, and PIN4, a member of the parvulin family of peptidylprolyl cis/trans isomerases that promotes mitosis and regulates cell proliferation, were found consistently decreased in PBMCs from PBC individuals in discordant monozygotic twins.
      • Mitchell M.M.
      • Lleo A.
      • Zammataro L.
      • Mayo M.J.
      • Invernizzi P.
      • Bach N.
      • et al.
      Epigenetic investigation of variably X chromosome inactivated genes in monozygotic female twins discordant for primary biliary cirrhosis.
      However, although partial and variable methylation on CpG sites was evident, a correlation with transcript levels was not observed. Furthermore, in CD4+ T cells from patients with PBC, a negative correlation was evident between CD40L promoter hypomethylation and CD40L expression levels.
      • Lleo A.
      • Liao J.
      • Invernizzi P.
      • Zhao M.
      • Bernuzzi F.
      • Ma L.
      • et al.
      Immunoglobulin M levels inversely correlate with CD40 ligand promoter methylation in patients with primary biliary cirrhosis.
      CD40L hypomethylation was also associated with increased serum IgM levels.
      • Lleo A.
      • Liao J.
      • Invernizzi P.
      • Zhao M.
      • Bernuzzi F.
      • Ma L.
      • et al.
      Immunoglobulin M levels inversely correlate with CD40 ligand promoter methylation in patients with primary biliary cirrhosis.
      Later on, a genome-wide analysis was able to identify consistent methylation profile alterations in 60 different gene regions from PBMCs of PBC individuals.
      • Selmi C.
      • Cavaciocchi F.
      • Lleo A.
      • Cheroni C.
      • De Francesco R.
      • Lombardi S.A.
      • et al.
      Genome-wide analysis of DNA methylation, copy number variation, and gene expression in monozygotic twins discordant for primary biliary cirrhosis.
      Of note, all these regions were hypermethylated in the affected twins, compared with the non-affected twins, and 51 genes were specifically mapped to the X chromosome while only nine genes were found in autosomal chromosomes. Notably, some of the genes that were found to be hypermethylated code for proteins involved in bile salt and ion transport. Preponderant methylation disturbances linked to the X chromosome were also found in another study in which the authors analysed methylation profiles on the X chromosome, reporting hypermethylation of FUNDC2 (involved in mitophagy) in CD8+ T cells from patients with PBC, while the chemokine receptor CXCR3 was demethylated in CD4+ T cells.
      • Lleo A.
      • Zhang W.
      • Zhao M.
      • Tan Y.
      • Bernuzzi F.
      • Zhu B.
      • et al.
      DNA methylation profiling of the X chromosome reveals an aberrant demethylation on CXCR3 promoter in primary biliary cirrhosis.
      Table 1Abnormalities in DNA methylation and histone post-translational modifications in PBC.
      DNA methylationComparisonPromoter methylationSampleInverse correlation with gene expressionRef.
      CLIC2, PIN4Discordant (n = 4) and concordant (n = 1) monozygotic twinsVariableSerumNo
      • Mitchell M.M.
      • Lleo A.
      • Zammataro L.
      • Mayo M.J.
      • Invernizzi P.
      • Bach N.
      • et al.
      Epigenetic investigation of variably X chromosome inactivated genes in monozygotic female twins discordant for primary biliary cirrhosis.
      CD40LPBC (n = 20) vs. healthy controls (n = 20)HypoSerum

      (CD4+ cells)
      Yes
      • Lleo A.
      • Liao J.
      • Invernizzi P.
      • Zhao M.
      • Bernuzzi F.
      • Ma L.
      • et al.
      Immunoglobulin M levels inversely correlate with CD40 ligand promoter methylation in patients with primary biliary cirrhosis.
      SMARCA1PBC monozygotic twins (n = 3) vs. discordant sisters (n = 8)HyperSerum

      (PBMCs)
      Yes
      • Selmi C.
      • Cavaciocchi F.
      • Lleo A.
      • Cheroni C.
      • De Francesco R.
      • Lombardi S.A.
      • et al.
      Genome-wide analysis of DNA methylation, copy number variation, and gene expression in monozygotic twins discordant for primary biliary cirrhosis.
      ABCD1, ATP12A, ATP5A1, BCAP31, BGN, BRCC3, CFP, CHST7, CTAG1A, CTAG1B, DDX41, FAM104B, FGD1, FUNDC2, GAGE 12B, 12I, 2A, 5, 7,8, GTPBP6,

      HCCS, HOXD4, IDH3G, IDS, IRAK1, KBTBD6, MAGEA3, MAGEA6, MAGEA9, MAGED4B, MTCP1, MTM1, MTMR8, NHS, ORC1L, CDK16, PDZD4, PHF16, PRKX, PRPF38A, RIBC1, RNF128, SCLY, SHROOM4, SLC10A3, SLC9A6, SLITRK2, SLITRK4, SSR4, TAF9B, TCEAL6, TUSC3, UBL4A, VCX2, VCX3A, YIPF6, ZIC3, ZNF182
      PBC monozygotic twins (n = 3) vs. discordant sisters (n = 8)HyperSerum

      (PBMCs)
      No
      • Selmi C.
      • Cavaciocchi F.
      • Lleo A.
      • Cheroni C.
      • De Francesco R.
      • Lombardi S.A.
      • et al.
      Genome-wide analysis of DNA methylation, copy number variation, and gene expression in monozygotic twins discordant for primary biliary cirrhosis.
      FUNDC2PBC (n = 30) vs. healthy controls (n = 30)HyperSerum

      (CD8+ cells)
      Yes
      • Lleo A.
      • Zhang W.
      • Zhao M.
      • Tan Y.
      • Bernuzzi F.
      • Zhu B.
      • et al.
      DNA methylation profiling of the X chromosome reveals an aberrant demethylation on CXCR3 promoter in primary biliary cirrhosis.
      CXCR3PBC (n = 30) vs. healthy controls (n = 30)HypoSerum

      (CD4+ cells)
      Yes
      • Lleo A.
      • Zhang W.
      • Zhao M.
      • Tan Y.
      • Bernuzzi F.
      • Zhu B.
      • et al.
      DNA methylation profiling of the X chromosome reveals an aberrant demethylation on CXCR3 promoter in primary biliary cirrhosis.
      UBE2APBC (n = 30) vs. healthy controls (n = 30)HyperSerum

      (CD4+ cells)
      No
      • Lleo A.
      • Zhang W.
      • Zhao M.
      • Tan Y.
      • Bernuzzi F.
      • Zhu B.
      • et al.
      DNA methylation profiling of the X chromosome reveals an aberrant demethylation on CXCR3 promoter in primary biliary cirrhosis.
      IL1RAPL2,PBC (n = 30) vs. healthy controls (n = 30)HyperSerum

      (CD14+ cells)
      Not expressed
      • Lleo A.
      • Zhang W.
      • Zhao M.
      • Tan Y.
      • Bernuzzi F.
      • Zhu B.
      • et al.
      DNA methylation profiling of the X chromosome reveals an aberrant demethylation on CXCR3 promoter in primary biliary cirrhosis.
      SHROOM2, ATP6AP2, KIAA1210, RAP2C, HTATSF1, SRPK3, IDH3GPBC (n = 30) vs. healthy controls (n = 30)HypoSerum

      (CD4+ cells)
      Not measured
      • Lleo A.
      • Zhang W.
      • Zhao M.
      • Tan Y.
      • Bernuzzi F.
      • Zhu B.
      • et al.
      DNA methylation profiling of the X chromosome reveals an aberrant demethylation on CXCR3 promoter in primary biliary cirrhosis.
      SH3KBP1, PHEX, USP9X, EBP, PQBP1, KDM5C, IL13RA1, CUL4B, DCAF12L1, GPC4, FAM50APBC (n = 30) vs. healthy controls (n = 30)HyperSerum

      (CD4+ cells)
      Not measured
      • Lleo A.
      • Zhang W.
      • Zhao M.
      • Tan Y.
      • Bernuzzi F.
      • Zhu B.
      • et al.
      DNA methylation profiling of the X chromosome reveals an aberrant demethylation on CXCR3 promoter in primary biliary cirrhosis.
      MAGEB2, PIN4, IDH3GPBC (n = 30) vs. healthy controls (n = 30)HypoSerum

      (CD8+ cells)
      Not measured
      • Lleo A.
      • Zhang W.
      • Zhao M.
      • Tan Y.
      • Bernuzzi F.
      • Zhu B.
      • et al.
      DNA methylation profiling of the X chromosome reveals an aberrant demethylation on CXCR3 promoter in primary biliary cirrhosis.
      GPR143, CHST7, NDUFB11, RBM10, ZXDB, DOCK11, IL13RA1, RNF113A, AIFM1, MPP1, RAB39BPBC (n = 30) vs. healthy controls (n = 30)HyperSerum

      (CD8+ cells)
      Not measured
      • Lleo A.
      • Zhang W.
      • Zhao M.
      • Tan Y.
      • Bernuzzi F.
      • Zhu B.
      • et al.
      DNA methylation profiling of the X chromosome reveals an aberrant demethylation on CXCR3 promoter in primary biliary cirrhosis.
      SYAP1, DYNLT3, CASK, GPR173, CITED1, GPRASP1, RNF128, NSDHL, SSR4PBC (n = 30) vs. healthy controls (n = 30)HypoSerum

      (CD14+ cells)
      Not measured
      • Lleo A.
      • Zhang W.
      • Zhao M.
      • Tan Y.
      • Bernuzzi F.
      • Zhu B.
      • et al.
      DNA methylation profiling of the X chromosome reveals an aberrant demethylation on CXCR3 promoter in primary biliary cirrhosis.
      KAL1, SH3KBP1, USP11, PIM2, PCDH11X, PSMD10, ATG4A, DCAF12L1, FUNDC2PBC (n = 30) vs. healthy controls (n = 30)HyperSerum

      (CD14+ cells)
      Not measured
      • Lleo A.
      • Zhang W.
      • Zhao M.
      • Tan Y.
      • Bernuzzi F.
      • Zhu B.
      • et al.
      DNA methylation profiling of the X chromosome reveals an aberrant demethylation on CXCR3 promoter in primary biliary cirrhosis.
      Histone post-translational regulatorsComparisonExpressionSampleFunctionRef.
      β-arrestin 1PBC (n = 60) vs. HBV-infected patients (n = 60)UpAutoreactive T cellsHistone H4 acetylation
      • Hu Z.
      • Huang Y.
      • Liu Y.
      • Sun Y.
      • Zhou Y.
      • Gu M.
      • et al.
      Beta-Arrestin 1 modulates functions of autoimmune T cells from primary biliary cirrhosis patients.
      HBV, hepatitis B virus; PBC, primary biliary cholangitis; PBMC, peripheral blood mononuclear cells.

      Histone variants and post-translational modifications

      Histones are regarded as the major subunits of nucleosomes. They can be subdivided into core (H2A, H2B, H3 and H4) and linker (H1 and H5) histones.
      • Cedar H.
      • Bergman Y.
      Linking DNA methylation and histone modification: patterns and paradigms.
      These components may assemble into octamers, wrapped by DNA, and may be post-translationally modified to control the accessibility of the transcriptional machinery to specific genes. Acetylation, methylation, phosphorylation, ubiquitination and sumoylation represent the majority of modifications found in histone proteins.
      • Freitas M.A.
      • Sklenar A.R.
      • Parthun M.R.
      Application of mass spectrometry to the identification and quantification of histone post-translational modifications.
      These dynamic processes are controlled by several modifying enzymes, including histone methyltransferases and demethylases and histone acetyltransferases and deacetylases.
      • Kouzarides T.
      Chromatin modifications and their function.
      Although there has been limited research on the impact of histone modifications in PBC, β-arrestin 1, a protein that promotes histone H4 acetylation in CD4+ cells, was increased in T lymphocytes from patients with PBC, positively correlating with PBC severity.
      • Hu Z.
      • Huang Y.
      • Liu Y.
      • Sun Y.
      • Zhou Y.
      • Gu M.
      • et al.
      Beta-Arrestin 1 modulates functions of autoimmune T cells from primary biliary cirrhosis patients.
      Notably, β-arrestin 1 overexpression increased T cell proliferation and altered the expression of several genes involved in autoimmunity, increasing histone H4 acetylation at the promoter regions of CD40L, LIGHT, IL17 and IFNG genes, which were previously shown to be deregulated in patients with PBC, while histone H4 acetylation was decreased in the promoter regions of TRAIL, APO2 and HDAC7A.
      Epigenetic abnormalities, including alterations in DNA methylation, histone modifications and miRNA disturbances, are characteristic of patients with PBC, and may significantly impact on aetiopathogenesis.

      MicroRNAs

      MicroRNAs (miRNAs or miRs) represent one of the most studied epigenetic elements in humans. Disturbances in miR expression profiles have been linked to a wide range of human disorders, including liver diseases,
      • Szabo G.
      • Bala S.
      MicroRNAs in liver disease.
      cholangiopathies,
      • Esparza-Baquer A.
      • Labiano I.
      • Bujanda L.
      • Perugorria M.J.
      • Banales J.M.
      MicroRNAs in cholangiopathies: potential diagnostic and therapeutic tools.
      • Olaizola P.
      • Lee-Law P.Y.
      • Arbelaiz A.
      • Lapitz A.
      • Perugorria M.J.
      • Bujanda L.
      • et al.
      MicroRNAs and extracellular vesicles in cholangiopathies.
      and autoimmune syndromes,
      • Brooks W.H.
      • Le Dantec C.
      • Pers J.O.
      • Youinou P.
      • Renaudineau Y.
      Epigenetics and autoimmunity.
      where hundreds of miRs were shown to be differentially expressed. In particular, microarray analysis of liver, sera and PBMCs from patients with PBC revealed more than 200 differentially expressed miRs. Some of them have been proposed as non-invasive biomarkers of disease, whereas miR-506 has arisen as a key player in PBC cholangiocyte pathobiology.

      MiR-506 & AE2

      In a microarray analysis comparing the expression of 377 miRs in PBC livers and healthy controls, 35 miRs were found to be dysregulated in PBC.
      • Padgett K.A.
      • Lan R.Y.
      • Leung P.C.
      • Lleo A.
      • Dawson K.
      • Pfeiff J.
      • et al.
      Primary biliary cirrhosis is associated with altered hepatic microRNA expression.
      Among them, hsa-miR-506-3p (miR-506) stood out as a potential direct regulator of AE2 expression because of its upregulation in PBC livers and partial complementarity to a human specific AE2-UTR-3′ mRNA sequence. The upregulation of miR-506 in PBC livers compared to healthy controls was confirmed by quantitative PCR (qPCR) in an independent cohort of patients.
      • Banales J.M.
      • Saez E.
      • Uriz M.
      • Sarvide S.
      • Urribarri A.D.
      • Splinter P.
      • et al.
      Up-regulation of microRNA 506 leads to decreased Cl−/HCO3− anion exchanger 2 expression in biliary epithelium of patients with primary biliary cirrhosis.
      In situ hybridization showed that miR-506 was specifically concentrated in intrahepatic biliary cells, while there was negligible miR-506 expression in control individuals (i.e., healthy or primary sclerosing cholangitis livers).
      • Banales J.M.
      • Saez E.
      • Uriz M.
      • Sarvide S.
      • Urribarri A.D.
      • Splinter P.
      • et al.
      Up-regulation of microRNA 506 leads to decreased Cl−/HCO3− anion exchanger 2 expression in biliary epithelium of patients with primary biliary cirrhosis.
      • Ananthanarayanan M.
      • Banales J.M.
      • Guerra M.T.
      • Spirli C.
      • Munoz-Garrido P.
      • Mitchell-Richards K.
      • et al.
      Post-translational regulation of the type III inositol 1,4,5-trisphosphate receptor by miRNA-506.
      Molecular studies in cultured human cholangiocytes demonstrated that miR-506 directly binds to the predicted (in silico) binding site of AE2 mRNA, resulting in decreased translation to protein, impaired Na+-independent Cl/HCO3exchange activity and reduced secretin-stimulated apical hydroionic fluxes, strongly suggesting a pivotal role of the miR-506/AE2 axis in the regulation of pH homeostasis and the biliary bicarbonate secretory system in PBC. Moreover, miR-506 was also found upregulated, by qPCR, in cholangiocytes isolated from PBC livers compared to normal human cholangiocytes in culture, and the molecular targeting of this miR with complementary anti-sense oligonucleotides restored the characteristic impaired AE2 activity of PBC cholangiocytes,
      • Melero S.
      • Spirli C.
      • Zsembery A.
      • Medina J.F.
      • Joplin R.E.
      • Duner E.
      • et al.
      Defective regulation of cholangiocyte Cl−/HCO3(−) and Na+/H+ exchanger activities in primary biliary cirrhosis.
      highlighting the potential therapeutic value of miR-506 inhibition in PBC.
      MiR-506 is upregulated in cholangiocytes from patients with PBC and directly targets AE2 mRNA, promoting the development of PBC-like features in cholangiocytes and immune activation.
      Thereafter, the molecular mechanisms regulating the miR-506 gene promoter activity and the effect of this miR on cholangiocyte pathobiology and immune activation was further investigated.
      • Erice O.
      • Munoz-Garrido P.
      • Vaquero J.
      • Perugorria M.J.
      • Fernandez-Barrena M.G.
      • Saez E.
      • et al.
      MicroRNA-506 promotes primary biliary cholangitis-like features in cholangiocytes and immune activation.
      Several pro-inflammatory cytokines (i.e., IL-8, IL-12, IL-17, IL-18 and TNF-α) usually found elevated in PBC livers were able to stimulate the miR-506 promoter activity, whereas different pro-fibrogenic cytokines, bile acids, oestrogens, glucocorticoids or growth factors had no effect. On the other hand, experimental overexpression of miR-506 in cultured human cholangiocytes altered the expression of several proteins involved in mitochondrial energetic metabolism, resulting in increased mitochondrial baseline respiration, maximal respiration, ATP-linked respiration and non-mitochondrial respiration, coupled with greater extracellular acidification rate, glycolysis and oxidative phosphorylation processes, when compared to control cells. However, increased proton leak and mitochondrial uncoupling were also associated with miR-506 overexpression, which in the end resulted in decreased ATP levels because of dysfunctional mitochondrial activity. In addition, pro-inflammatory, pro-fibrotic, mesenchymal and senescence markers were found upregulated in miR-506-overexpressing cholangiocytes compared to controls, resulting in reduced cholangiocyte adhesion and migration, as well as increased cellular stress that sensitises cholangiocytes to the apoptotic effect of toxic bile acids. Notably, miR-506-overexpressing cholangiocytes were also characterised by robust and aberrant overexpression of the PDC-E2 protein, mainly localised at the cytoplasm and plasma membrane, suggesting a potential link between miR-506, AMAs and autoimmune phenomena in PBC. In order to investigate this hypothesis, PBMCs from patients with PBC were co-cultured with miR-506-overexpressing cholangiocytes, resulting in higher numbers of lymphocyte aggregates, characterised by increased proliferation and activation compared with control conditions (i.e., PBMCs from patients with PBC co-cultured with control cholangiocytes). These data are consistent with previous results indicating that cholangiocytes from patients with PBC exhibit increased expression of major histocompatibility complex class I and II molecules, in parallel with ICAM-1 upregulation,
      • Tsuneyama K.
      • Van de Water J.
      • Leung P.S.
      • Cha S.
      • Nakanuma Y.
      • Kaplan M.
      • et al.
      Abnormal expression of the E2 component of the pyruvate dehydrogenase complex on the luminal surface of biliary epithelium occurs before major histocompatibility complex class II and BB1/B7 expression.
      • Ayres R.C.
      • Neuberger J.M.
      • Shaw J.
      • Joplin R.
      • Adams D.H.
      Intercellular adhesion molecule-1 and MHC antigens on human intrahepatic bile duct cells: effect of pro-inflammatory cytokines.
      • Nishimoto H.
      • Yamada G.
      • Mizuno M.
      • Tsuji T.
      Immunoelectron microscopic localization of MHC class 1 and 2 antigens on bile duct epithelial cells in patients with primary biliary cirrhosis.
      • Yokomori H.
      • Oda M.
      • Yoshimura K.
      • Nomura M.
      • Ogi M.
      • Wakabayashi G.
      • et al.
      Expression of intercellular adhesion molecule-1 and lymphocyte function-associated antigen-1 protein and messenger RNA in primary biliary cirrhosis.
      suggesting an important role for these diseased cholangiocytes in the promotion of autoimmunity.

      MiR-506 & InsP3R3

      Besides AE2, another direct target of miR-506 with significant relevance in PBC pathogenesis is inositol 1,4,5-trisphosphate receptor 3 (InsP3R3, also called ITPR3), which functions as a calcium (Ca2+) release channel at the endoplasmic reticulum (ER) membrane of cholangiocytes. Under physiological conditions, acetylcholine raises the intracellular InsP3 levels in cholangiocytes, which via InsP3R3 interaction leads to increased cytoplasmic Ca2+ levels that further stimulate apical Cl secretion through the Ca2+-activated Cl channel transmembrane protein 16A (TMEM16A), ultimately resulting in bicarbonate secretion via AE2.
      • Minagawa N.
      • Nagata J.
      • Shibao K.
      • Masyuk A.I.
      • Gomes D.A.
      • Rodrigues M.A.
      • et al.
      Cyclic AMP regulates bicarbonate secretion in cholangiocytes through release of ATP into bile.
      • Hirata K.
      • Nathanson M.H.
      Bile duct epithelia regulate biliary bicarbonate excretion in normal rat liver.
      In PBC, InsP3R3 expression is downregulated in cholangiocytes,
      • Shibao K.
      • Hirata K.
      • Robert M.E.
      • Nathanson M.H.
      Loss of inositol 1,4,5-trisphosphate receptors from bile duct epithelia is a common event in cholestasis.
      resulting in impaired intracellular Ca2+ signalling and biliary bicarbonate secretion, thus triggering cholestasis. InsP3R3 mRNA contains two highly-conserved miR-506 binding sites that regulate its expression.
      • Ananthanarayanan M.
      • Banales J.M.
      • Guerra M.T.
      • Spirli C.
      • Munoz-Garrido P.
      • Mitchell-Richards K.
      • et al.
      Post-translational regulation of the type III inositol 1,4,5-trisphosphate receptor by miRNA-506.
      Thus, miR-506-overexpressing cholangiocytes were characterised by decreased InsP3R3 mRNA and protein levels, leading to a significant reduction in Ca2+ release from the ER and biliary secretory failure.
      • Banales J.M.
      • Saez E.
      • Uriz M.
      • Sarvide S.
      • Urribarri A.D.
      • Splinter P.
      • et al.
      Up-regulation of microRNA 506 leads to decreased Cl−/HCO3− anion exchanger 2 expression in biliary epithelium of patients with primary biliary cirrhosis.
      • Ananthanarayanan M.
      • Banales J.M.
      • Guerra M.T.
      • Spirli C.
      • Munoz-Garrido P.
      • Mitchell-Richards K.
      • et al.
      Post-translational regulation of the type III inositol 1,4,5-trisphosphate receptor by miRNA-506.

      Other dysregulated miRNAs in PBC pathogenesis

      New evidence indicates that other miRs may impact on the development and progression of PBC (Table 2). Among the miRs found to be dysregulated in PBC livers by microarray analysis, qPCR confirmed that miR-328 and miR-299-5p were upregulated while miR-122a and miR26a were downregulated.
      • Padgett K.A.
      • Lan R.Y.
      • Leung P.C.
      • Lleo A.
      • Dawson K.
      • Pfeiff J.
      • et al.
      Primary biliary cirrhosis is associated with altered hepatic microRNA expression.
      Their predicted targets were related to the regulation of cell proliferation, apoptosis, inflammation, oxidative stress and metabolism. In particular, miR-26a specifically targets polycomb group protein EZH2, a protein known to contribute to cholangiocyte senescence in PBC.
      • Sasaki M.
      • Ikeda H.
      • Sato Y.
      • Nakanuma Y.
      Decreased expression of Bmi1 is closely associated with cellular senescence in small bile ducts in primary biliary cirrhosis.
      • Sander S.
      • Bullinger L.
      • Klapproth K.
      • Fiedler K.
      • Kestler H.A.
      • Barth T.F.
      • et al.
      MYC stimulates EZH2 expression by repression of its negative regulator miR-26a.
      On the other hand, miR-21 was found markedly increased in PBC livers, contributing to regulated necrosis (i.e., necroptosis), also affecting cholangiocytes.
      • Afonso M.B.
      • Rodrigues P.M.
      • Simao A.L.
      • Gaspar M.M.
      • Carvalho T.
      • Borralho P.
      • et al.
      MiRNA-21 ablation protects against liver injury and necroptosis in cholestasis.
      • Afonso M.B.
      • Rodrigues P.M.
      • Simao A.L.
      • Ofengeim D.
      • Carvalho T.
      • Amaral J.D.
      • et al.
      Activation of necroptosis in human and experimental cholestasis.
      MiR-21−/− mice subjected to experimental obstructive cholestasis (i.e., bile duct ligation) displayed reduced serum levels of ALP, bilirubin and bile acids, indicating its role in the regulation of bile acid metabolism. Also, the expression levels of FXR and some bile acid transporters, including organic anion-transporting polypeptide (Oatp), sodium/taurocholate co-transporting polypeptide (Ntcp), multispecific organic anion transporter 3 (Mrp3) and bile salt export pump (Bsep), were upregulated in miR-21−/− mice, but the exact mechanism behind this choleretic effect should be further investigated. Moreover, miR-21 was also linked to immune cell activation in PBC, as dnTGF-βRII mice presented higher miR-21 expression levels in hepatic CD44+ CD62L effector CD8+ T cells when compared with corresponding cells from the spleen and mesenteric lymph nodes.
      • Ando Y.
      • Yang G.X.
      • Kenny T.P.
      • Kawata K.
      • Zhang W.
      • Huang W.
      • et al.
      Overexpression of microRNA-21 is associated with elevated pro-inflammatory cytokines in dominant-negative TGF-beta receptor type II mouse.
      Experimental overexpression of miR-21 in B6 CD8+ T cells increased the production of pro-inflammatory IFN-γ and IL-17 cytokines, and hampered apoptotic cell death of autoreactive CD8+ cells, thus suggesting a potential role of miR-21 in the promotion of chronic nonsuppurative cholangitis. MiR-155, which is related to immune cell activation mainly through direct targeting of suppressor of cytokine signalling 1 (SOCS1), a well-known antagonist of inflammation, was also found to be overexpressed in PBC livers.
      • Kempinska-Podhorodecka A.
      • Milkiewicz M.
      • Wasik U.
      • Ligocka J.
      • Zawadzki M.
      • Krawczyk M.
      • et al.
      Decreased expression of vitamin D receptor affects an immune response in primary biliary cholangitis via the VDR-miRNA155-SOCS1 pathway.
      Finally, miR-34a and miR-132 were shown to be increased in the liver of cirrhotic patients with PBC when compared to healthy individuals,
      • Wasik U.
      • Milkiewicz M.
      • Kempinska-Podhorodecka A.
      • Milkiewicz P.
      Protection against oxidative stress mediated by the Nrf2/Keap1 axis is impaired in Primary Biliary Cholangitis.
      which might be related with oxidative stress by direct targeting NRF2, and thus triggering disease progression.
      Table 2MiRs differentially expressed in the liver of patients with PBC.
      miRNAComparisonExpressionValidated targetFunctionRef.
      miR-122-5pPBC (n = 6) vs. healthy controls (n = 5)DownCell proliferation, senescence, apoptosis, inflammation, oxidative stress and metabolism
      • Padgett K.A.
      • Lan R.Y.
      • Leung P.C.
      • Lleo A.
      • Dawson K.
      • Pfeiff J.
      • et al.
      Primary biliary cirrhosis is associated with altered hepatic microRNA expression.
      miR-26a-5pDown
      • Padgett K.A.
      • Lan R.Y.
      • Leung P.C.
      • Lleo A.
      • Dawson K.
      • Pfeiff J.
      • et al.
      Primary biliary cirrhosis is associated with altered hepatic microRNA expression.
      miR-328-3pUp
      • Padgett K.A.
      • Lan R.Y.
      • Leung P.C.
      • Lleo A.
      • Dawson K.
      • Pfeiff J.
      • et al.
      Primary biliary cirrhosis is associated with altered hepatic microRNA expression.
      miR-299-5pUp
      • Padgett K.A.
      • Lan R.Y.
      • Leung P.C.
      • Lleo A.
      • Dawson K.
      • Pfeiff J.
      • et al.
      Primary biliary cirrhosis is associated with altered hepatic microRNA expression.
      miR-506-3pPBC (n = 6) vs. healthy cholangiocytes (n = 6)UpAE2Biliary bicarbonate secretion and pHi homeostasis
      • Banales J.M.
      • Saez E.
      • Uriz M.
      • Sarvide S.
      • Urribarri A.D.
      • Splinter P.
      • et al.
      Up-regulation of microRNA 506 leads to decreased Cl−/HCO3− anion exchanger 2 expression in biliary epithelium of patients with primary biliary cirrhosis.
      • Ananthanarayanan M.
      • Banales J.M.
      • Guerra M.T.
      • Spirli C.
      • Munoz-Garrido P.
      • Mitchell-Richards K.
      • et al.
      Post-translational regulation of the type III inositol 1,4,5-trisphosphate receptor by miRNA-506.
      PBC (n = 5) vs. healthy controls (n = 5)InsR3P3
      • Banales J.M.
      • Saez E.
      • Uriz M.
      • Sarvide S.
      • Urribarri A.D.
      • Splinter P.
      • et al.
      Up-regulation of microRNA 506 leads to decreased Cl−/HCO3− anion exchanger 2 expression in biliary epithelium of patients with primary biliary cirrhosis.
      • Ananthanarayanan M.
      • Banales J.M.
      • Guerra M.T.
      • Spirli C.
      • Munoz-Garrido P.
      • Mitchell-Richards K.
      • et al.
      Post-translational regulation of the type III inositol 1,4,5-trisphosphate receptor by miRNA-506.
      miR-21-5pPBC (n = 5) vs. healthy controls (n = 10)UpCDK2AP1Regulated necrosis
      • Afonso M.B.
      • Rodrigues P.M.
      • Simao A.L.
      • Gaspar M.M.
      • Carvalho T.
      • Borralho P.
      • et al.
      MiRNA-21 ablation protects against liver injury and necroptosis in cholestasis.
      miR-155PBC (n = 16) vs. healthy controls (n = 11)UpSOCS1Immune cell activation
      • Kempinska-Podhorodecka A.
      • Milkiewicz M.
      • Wasik U.
      • Ligocka J.
      • Zawadzki M.
      • Krawczyk M.
      • et al.
      Decreased expression of vitamin D receptor affects an immune response in primary biliary cholangitis via the VDR-miRNA155-SOCS1 pathway.
      miR-132, miR-34aCirrhotic PBC (n = 15) vs. healthy controls (n = 10)UpNRF2Oxidative stress
      • Wasik U.
      • Milkiewicz M.
      • Kempinska-Podhorodecka A.
      • Milkiewicz P.
      Protection against oxidative stress mediated by the Nrf2/Keap1 axis is impaired in Primary Biliary Cholangitis.
      PBC, primary biliary cholangitis.

      MiRNAs as diagnostic and prognostic markers

      Alterations in the expression of circulating miRs have been reported in PBC, and are postulated as potential diagnostic and/or prognostic non-invasive biomarkers of disease (Table 3, Table 4).
      Table 3MiRs differentially expressed in serum of patients with PBC.
      miRNAComparisonExpressionClinical correlationsAUCSensitivitySpecificityRef.
      miR-122-5pPBC (n = 3) vs. healthy controls (n = 3)Up0.9050.8050.883
      • Tan Y.
      • Pan T.
      • Ye Y.
      • Ge G.
      • Chen L.
      • Wen D.
      • et al.
      Serum microRNAs as potential biomarkers of primary biliary cirrhosis.
      miR-141-3pUp
      • Tan Y.
      • Pan T.
      • Ye Y.
      • Ge G.
      • Chen L.
      • Wen D.
      • et al.
      Serum microRNAs as potential biomarkers of primary biliary cirrhosis.
      miR-26b-5pDown
      • Tan Y.
      • Pan T.
      • Ye Y.
      • Ge G.
      • Chen L.
      • Wen D.
      • et al.
      Serum microRNAs as potential biomarkers of primary biliary cirrhosis.
      miR-34a-5pUp0.6620.6670.689
      • Tan Y.
      • Pan T.
      • Ye Y.
      • Ge G.
      • Chen L.
      • Wen D.
      • et al.
      Serum microRNAs as potential biomarkers of primary biliary cirrhosis.
      miR-27b-3pUp0.5710.3040.867
      • Tan Y.
      • Pan T.
      • Ye Y.
      • Ge G.
      • Chen L.
      • Wen D.
      • et al.
      Serum microRNAs as potential biomarkers of primary biliary cirrhosis.
      miR-505-3pPBC (n = 10) vs. healthy controls (n = 5)Down
      • Ninomiya M.
      • Kondo Y.
      • Funayama R.
      • Nagashima T.
      • Kogure T.
      • Kakazu E.
      • et al.
      Distinct microRNAs expression profile in primary biliary cirrhosis and evaluation of miR 505-3p and miR197-3p as novel biomarkers.
      miR-197-3pDown
      • Ninomiya M.
      • Kondo Y.
      • Funayama R.
      • Nagashima T.
      • Kogure T.
      • Kakazu E.
      • et al.
      Distinct microRNAs expression profile in primary biliary cirrhosis and evaluation of miR 505-3p and miR197-3p as novel biomarkers.
      miR-500a-3pDown
      • Ninomiya M.
      • Kondo Y.
      • Funayama R.
      • Nagashima T.
      • Kogure T.
      • Kakazu E.
      • et al.
      Distinct microRNAs expression profile in primary biliary cirrhosis and evaluation of miR 505-3p and miR197-3p as novel biomarkers.
      miR-122PBC UDCA non-responders (n = 5) vs. PBC UDCA responders (n = 15)Up↑ Bilirubin, AST and ALT
      • Sakamoto T.
      • Morishita A.
      • Nomura T.
      • Tani J.
      • Miyoshi H.
      • Yoneyama H.
      • et al.
      Identification of microRNA profiles associated with refractory primary biliary cirrhosis.
      miR-378fUp↑ AST, ALT and

      γ-guanosine triphosphate
      • Sakamoto T.
      • Morishita A.
      • Nomura T.
      • Tani J.
      • Miyoshi H.
      • Yoneyama H.
      • et al.
      Identification of microRNA profiles associated with refractory primary biliary cirrhosis.
      miR-4311Down↓ AST and ALT
      • Sakamoto T.
      • Morishita A.
      • Nomura T.
      • Tani J.
      • Miyoshi H.
      • Yoneyama H.
      • et al.
      Identification of microRNA profiles associated with refractory primary biliary cirrhosis.
      miR-4714-3pDown↓ Bilirubin and LDH
      • Sakamoto T.
      • Morishita A.
      • Nomura T.
      • Tani J.
      • Miyoshi H.
      • Yoneyama H.
      • et al.
      Identification of microRNA profiles associated with refractory primary biliary cirrhosis.
      miR-139-5pPBC with portal hypertension (n = 6) and PBC with hepatic failure (n = 6) vs. healthy controls (n = 6)Down↑ Disease severity
      • Tomiyama T.
      • Yang G.X.
      • Zhao M.
      • Zhang W.
      • Tanaka H.
      • Wang J.
      • et al.
      The modulation of co-stimulatory molecules by circulating exosomes in primary biliary cirrhosis.
      PBC, primary biliary cholangitis.
      Table 4MiRs differentially expressed in the PBMCs and EVs of patients with PBC.
      miRNAComparisonExpressionClinical correlationsRef.
      miR-299-5pPBC UDCA non-responders (n = 18) vs. healthy controls (n = 30)Up↑ ALP, GGT, Bilirubin and IgM
      • Katsushima F.
      • Takahashi A.
      • Sakamoto N.
      • Kanno Y.
      • Abe K.
      • Ohira H.
      Expression of micro-RNAs in peripheral blood mononuclear cells from primary biliary cirrhosis patients.
      miR-146a-5pPBC (n = 58) vs. healthy controls (n = 30)Up
      • Katsushima F.
      • Takahashi A.
      • Sakamoto N.
      • Kanno Y.
      • Abe K.
      • Ohira H.
      Expression of micro-RNAs in peripheral blood mononuclear cells from primary biliary cirrhosis patients.
      miR-155-5pUp
      • Katsushima F.
      • Takahashi A.
      • Sakamoto N.
      • Kanno Y.
      • Abe K.
      • Ohira H.
      Expression of micro-RNAs in peripheral blood mononuclear cells from primary biliary cirrhosis patients.
      miR-26aPBC (n = 58) vs. AIH (n = 25)Up↑ GGT
      • Katsushima F.
      • Takahashi A.
      • Sakamoto N.
      • Kanno Y.
      • Abe K.
      • Ohira H.
      Expression of micro-RNAs in peripheral blood mononuclear cells from primary biliary cirrhosis patients.
      miR-328Up↑ GGT
      • Katsushima F.
      • Takahashi A.
      • Sakamoto N.
      • Kanno Y.
      • Abe K.
      • Ohira H.
      Expression of micro-RNAs in peripheral blood mononuclear cells from primary biliary cirrhosis patients.
      Let-7aUp
      • Katsushima F.
      • Takahashi A.
      • Sakamoto N.
      • Kanno Y.
      • Abe K.
      • Ohira H.
      Expression of micro-RNAs in peripheral blood mononuclear cells from primary biliary cirrhosis patients.
      Let-7b-5pPBC (n = 60) vs. healthy controls (n = 60)Down↑ Disease severity stage, Mayo risk score, IL-18 and ALP
      • Qian C.
      • Chen S.X.
      • Ren C.L.
      • Zhong R.Q.
      • Deng A.M.
      Qin Q.
      miR-181a-5pPBC (n = 14) vs. healthy controls (n = 10)Down↑ Activation of CD4+ T cells
      • Nakagawa R.
      • Muroyama R.
      • Saeki C.
      • Goto K.
      • Kaise Y.
      • Koike K.
      • et al.
      MiR-425 regulates inflammatory cytokine production in CD4(+) T cells via N-Ras upregulation in primary biliary cholangitis.
      miR-181b-5pDown
      • Nakagawa R.
      • Muroyama R.
      • Saeki C.
      • Goto K.
      • Kaise Y.
      • Koike K.
      • et al.
      MiR-425 regulates inflammatory cytokine production in CD4(+) T cells via N-Ras upregulation in primary biliary cholangitis.
      miR-374bDown
      • Nakagawa R.
      • Muroyama R.
      • Saeki C.
      • Goto K.
      • Kaise Y.
      • Koike K.
      • et al.
      MiR-425 regulates inflammatory cytokine production in CD4(+) T cells via N-Ras upregulation in primary biliary cholangitis.
      miR-425Down
      • Nakagawa R.
      • Muroyama R.
      • Saeki C.
      • Goto K.
      • Kaise Y.
      • Koike K.
      • et al.
      MiR-425 regulates inflammatory cytokine production in CD4(+) T cells via N-Ras upregulation in primary biliary cholangitis.
      miR-223-3pPBC stage I (n = 17), stage II (n = 23), stage III (n = 16) and stage IV (n = 16) vs. healthy controls (n = 15)Down↑ Disease severity stage
      • Wang X.
      • Wen X.
      • Zhou J.
      • Qi Y.
      • Wu R.
      • Wang Y.
      • et al.
      MicroRNA-223 and microRNA-21 in peripheral blood B cells associated with progression of primary biliary cholangitis patients.
      miR-21-5pDown
      • Wang X.
      • Wen X.
      • Zhou J.
      • Qi Y.
      • Wu R.
      • Wang Y.
      • et al.
      MicroRNA-223 and microRNA-21 in peripheral blood B cells associated with progression of primary biliary cholangitis patients.
      miR-20a-5pPBC vs. healthy controlsDown
      • Qian C.
      • Wang H.Z.
      • Fan H.J.
      • Gu M.L.
      • Ren C.L.
      • Deng A.M.
      • et al.
      MicroRNA profiling in T cells of peripheral blood mononuclear cell from patients with primary biliary cirrhosis.
      miR-346Down
      • Qian C.
      • Wang H.Z.
      • Fan H.J.
      • Gu M.L.
      • Ren C.L.
      • Deng A.M.
      • et al.
      MicroRNA profiling in T cells of peripheral blood mononuclear cell from patients with primary biliary cirrhosis.
      miR-17-5pDown
      • Qian C.
      • Wang H.Z.
      • Fan H.J.
      • Gu M.L.
      • Ren C.L.
      • Deng A.M.
      • et al.
      MicroRNA profiling in T cells of peripheral blood mononuclear cell from patients with primary biliary cirrhosis.
      miR-451aUp
      • Qian C.
      • Wang H.Z.
      • Fan H.J.
      • Gu M.L.
      • Ren C.L.
      • Deng A.M.
      • et al.
      MicroRNA profiling in T cells of peripheral blood mononuclear cell from patients with primary biliary cirrhosis.
      miR-129-5pUp
      • Qian C.
      • Wang H.Z.
      • Fan H.J.
      • Gu M.L.
      • Ren C.L.
      • Deng A.M.
      • et al.
      MicroRNA profiling in T cells of peripheral blood mononuclear cell from patients with primary biliary cirrhosis.
      miR-15a-5pPBC (n = 9) vs. healthy controls (n = 9)Up
      • Qin B.
      • Huang F.
      • Liang Y.
      • Yang Z.
      • Zhong R.
      Analysis of altered microRNA expression profiles in peripheral blood mononuclear cells from patients with primary biliary cirrhosis.
      miR-20a-5pUp
      • Qin B.
      • Huang F.
      • Liang Y.
      • Yang Z.
      • Zhong R.
      Analysis of altered microRNA expression profiles in peripheral blood mononuclear cells from patients with primary biliary cirrhosis.
      miR-106b-5pUp
      • Qin B.
      • Huang F.
      • Liang Y.
      • Yang Z.
      • Zhong R.
      Analysis of altered microRNA expression profiles in peripheral blood mononuclear cells from patients with primary biliary cirrhosis.
      miR-140-3pUp
      • Qin B.
      • Huang F.
      • Liang Y.
      • Yang Z.
      • Zhong R.
      Analysis of altered microRNA expression profiles in peripheral blood mononuclear cells from patients with primary biliary cirrhosis.
      miR-181a-5pDown
      • Qin B.
      • Huang F.
      • Liang Y.
      • Yang Z.
      • Zhong R.
      Analysis of altered microRNA expression profiles in peripheral blood mononuclear cells from patients with primary biliary cirrhosis.
      miR-3654Down
      • Qin B.
      • Huang F.
      • Liang Y.
      • Yang Z.
      • Zhong R.
      Analysis of altered microRNA expression profiles in peripheral blood mononuclear cells from patients with primary biliary cirrhosis.
      miR-451aPBC (n = 29) vs. healthy controls (n = 30)Up
      • Tomiyama T.
      • Yang G.X.
      • Zhao M.
      • Zhang W.
      • Tanaka H.
      • Wang J.
      • et al.
      The modulation of co-stimulatory molecules by circulating exosomes in primary biliary cirrhosis.
      miR-642-3pUp
      • Tomiyama T.
      • Yang G.X.
      • Zhao M.
      • Zhang W.
      • Tanaka H.
      • Wang J.
      • et al.
      The modulation of co-stimulatory molecules by circulating exosomes in primary biliary cirrhosis.
      EVs, extracellular vesicles; PBC, primary biliary cholangitis; PBMC, peripheral blood mononuclear cells.
      In serum, miR-122-5p and miR-141-3p are increased in patients with PBC vs. healthy controls, while the levels of miR-26b-5p are reduced. This miRNA panel was associated with increased sensitivity, specificity and area under the receiver operating curve (AUC) for the diagnosis of PBC, when compared with both ALP or ANA serum levels.
      • Tan Y.
      • Pan T.
      • Ye Y.
      • Ge G.
      • Chen L.
      • Wen D.
      • et al.
      Serum microRNAs as potential biomarkers of primary biliary cirrhosis.
      Furthermore, deep sequencing analysis in the serum of patients with PBC revealed that miR-139-5p is downregulated and inversely correlated with the disease stage.
      • Katsumi T.
      • Ninomiya M.
      • Nishina T.
      • Mizuno K.
      • Tomita K.
      • Haga H.
      • et al.
      MiR-139-5p is associated with inflammatory regulation through c-FOS suppression, and contributes to the progression of primary biliary cholangitis.
      Reduced levels of this miRNA, along with miR-197-3p, miR-500a-3p and miR-505-3p were also evident in the serum of patients with PBC when compared to either healthy controls or patients with hepatitis B/C infection.
      • Ninomiya M.
      • Kondo Y.
      • Funayama R.
      • Nagashima T.
      • Kogure T.
      • Kakazu E.
      • et al.
      Distinct microRNAs expression profile in primary biliary cirrhosis and evaluation of miR 505-3p and miR197-3p as novel biomarkers.
      Importantly, changes in the expression of several miRs were reported in the serum of incomplete responders to UDCA vs. UDCA responders (up: miR-122 and miR-378; down: miR-4311 and miR-4714-3p), and have been postulated as prognostic biomarkers for PBC.
      • Sakamoto T.
      • Morishita A.
      • Nomura T.
      • Tani J.
      • Miyoshi H.
      • Yoneyama H.
      • et al.
      Identification of microRNA profiles associated with refractory primary biliary cirrhosis.
      Circulating miRNAs are potential non-invasive tools to predict prognosis and treatment response in patients with PBC.
      In PBMCs, an abnormal miR profile was identified in patients with PBC, which includes miR-181a, miR-181b, miR-374b and miR-425 downregulation in circulating CD4+ T cells. In particular, decreased miR-425 levels were associated with a pro-inflammatory phenotype.
      • Nakagawa R.
      • Muroyama R.
      • Saeki C.
      • Goto K.
      • Kaise Y.
      • Koike K.
      • et al.
      MiR-425 regulates inflammatory cytokine production in CD4(+) T cells via N-Ras upregulation in primary biliary cholangitis.
      The levels of let-7b were also reduced in PBMCs of patients with PBC and further correlated with disease severity,
      • Qian C.
      • Chen S.X.
      • Ren C.L.
      • Zhong R.Q.
      • Deng A.M.
      Qin Q.
      while miR-92a levels positively correlated with the frequency of IL-17-producing T helper cells.
      • Liang D.Y.
      • Hou Y.Q.
      • Luo L.J.
      • Ao L.
      Altered expression of miR-92a correlates with Th17 cell frequency in patients with primary biliary cirrhosis.
      In addition, miR-223-3p and miR-21-5p were downregulated, compared to healthy controls, in the PBMCs of patients with PBC and were inversely correlated with the disease staging.
      • Wang X.
      • Wen X.
      • Zhou J.
      • Qi Y.
      • Wu R.
      • Wang Y.
      • et al.
      MicroRNA-223 and microRNA-21 in peripheral blood B cells associated with progression of primary biliary cholangitis patients.
      Increased miR-299-5p levels in PBMCs were also found in inadequate responders to UDCA when compared to PBC responders and healthy controls, and positively correlated with ALP, GGT, bilirubin and IgM serum levels.
      • Katsushima F.
      • Takahashi A.
      • Sakamoto N.
      • Kanno Y.
      • Abe K.
      • Ohira H.
      Expression of micro-RNAs in peripheral blood mononuclear cells from primary biliary cirrhosis patients.
      Other miR alterations in PBMCs of patients with PBC,
      • Qian C.
      • Wang H.Z.
      • Fan H.J.
      • Gu M.L.
      • Ren C.L.
      • Deng A.M.
      • et al.
      MicroRNA profiling in T cells of peripheral blood mononuclear cell from patients with primary biliary cirrhosis.
      • Qin B.
      • Huang F.
      • Liang Y.
      • Yang Z.
      • Zhong R.
      Analysis of altered microRNA expression profiles in peripheral blood mononuclear cells from patients with primary biliary cirrhosis.
      but with unknown clinical correlation, are displayed in Table 4.
      Serum extracellular vesicles may also contain miRs that are potentially useful as biomarkers for PBC, as miR-451a and miR642-3p were increased in extracellular vesicles of PBC when compared with healthy individuals.
      • Tomiyama T.
      • Yang G.X.
      • Zhao M.
      • Zhang W.
      • Tanaka H.
      • Wang J.
      • et al.
      The modulation of co-stimulatory molecules by circulating exosomes in primary biliary cirrhosis.

      Long non-coding RNAs

      Long non-coding RNAs (lncRNAS) are highly-conserved RNA sequences (>200 nucleotides) that epigenetically regulate gene expression and a wide range of cellular processes.
      • Ponting C.P.
      • Oliver P.L.
      • Reik W.
      Evolution and functions of long noncoding RNAs.
      • Hung T.
      • Chang H.Y.
      Long noncoding RNA in genome regulation: prospects and mechanisms.
      • Mercer T.R.
      • Dinger M.E.
      • Mattick J.S.
      Long non-coding RNAs: insights into functions.
      Among the SNPs identified as increasing susceptibility to autoimmune diseases, ∼10% were mapped to lncRNAs and related with abnormal expression.
      • Ricano-Ponce I.
      • Wijmenga C.
      Mapping of immune-mediated disease genes.
      In PBC, 38 SNPs were mapped to lncRNAs.
      • Hrdlickova B.
      • Kumar V.
      • Kanduri K.
      • Zhernakova D.V.
      • Tripathi S.
      • Karjalainen J.
      • et al.
      Expression profiles of long non-coding RNAs located in autoimmune disease-associated regions reveal immune cell-type specificity.
      In addition, lncRNA AK053349 was found highly-expressed in CD8+ T cells and linked with autoimmunity and T lymphocyte activation.
      • Pang K.C.
      • Dinger M.E.
      • Mercer T.R.
      • Malquori L.
      • Grimmond S.M.
      • Chen W.
      • et al.
      Genome-wide identification of long noncoding RNAs in CD8+ T cells.
      LncRNA AK053349 expression was increased in PBMCs from patients with PBC and positively correlated with the Mayo risk score,
      • Zhang L.
      • Huang Y.
      • Wang H.
      • Kong W.
      • Ye X.
      • Chen Y.
      • et al.
      Increased expression of lncRNA AK053349 in peripheral blood mononuclear cells from patients with primary biliary cirrhosis and its clinical significance.
      underscoring its potential relevance in PBC pathogenesis and warranting further study.

      Conclusions and future directions

      The aetiopathogenesis of PBC is more complex than previously thought. The combination of genetic susceptibility, environmental exposures, and epigenetic alterations may contribute to disease development and progression. In this manuscript, the epigenetic alterations, mostly the ones related with AE2-linked secretory failure and cholestasis, were highlighted. So far, miR-506 has been identified as a potential contributor to PBC pathogenesis, promoting PBC-like features in cholangiocytes that subsequently stimulate immune activation (Fig. 1). MiR-506 impairs biliary bicarbonate secretion through direct targeting of both AE2 and InsR3P3, induces cholangiocyte dedifferentiation, stress and DNA damage, and promotes apoptosis induced by toxic bile acids. MiR-506 also impairs the mitochondrial energetic metabolism, induces PDC-E2 overexpression and mislocalisation, and promotes immune activation. This global overview, together with the observation that miR-506 molecular targeting results in the restoration of AE2 activity in PBC cholangiocytes in vitro, leads us to envision miR-506 as a key epigenetic player and potential therapeutic target in PBC. Since anti-miR strategies have been demonstrated as efficient and well-tolerated in patients with liver diseases like hepatitis C (i.e., anti-miR-122),
      • Sanchez-Nino M.D.
      • Ortiz A.
      HCV infection and miravirsen.
      • van der Ree M.H.
      • de Vree J.M.
      • Stelma F.
      • Willemse S.
      • van der Valk M.
      • Rietdijk S.
      • et al.
      Safety, tolerability, and antiviral effect of RG-101 in patients with chronic hepatitis C: a phase 1B, double-blind, randomised controlled trial.
      their efficacy for PBC should be determined. Moreover, future studies should determine the presence of circulating miR-506 and its potential prognostic value for PBC. The usefulness of circulating miRs as non-invasive biomarkers for PBC is currently under study.
      Figure thumbnail gr1
      Fig. 1MiR-506-induced secretory failure in PBC: A new hypothesis for disease pathogenesis. Under physiological conditions, secretin binds to its receptor in cholangiocytes, promoting the cAMP/PKA-dependent exocytosis of vesicles containing the Cl channel CFTR, AE2 and the water channel AQP1 to the apical membrane of cholangiocytes.
      • Tietz P.S.
      • LaRusso N.F.
      Cholangiocyte biology.
      • Tietz P.S.
      • Marinelli R.A.
      • Chen X.M.
      • Huang B.
      • Cohn J.
      • Kole J.
      • et al.
      Agonist-induced coordinated trafficking of functionally related transport proteins for water and ions in cholangiocytes.
      This leads to the Cl secretion via CFTR and its further exchange with HCO3 through AE2 that creates a luminal osmotic gradient for the movement of water by AQP1, consequently resulting in choleresis.
      • Banales J.M.
      • Prieto J.
      • Medina J.F.
      Cholangiocyte anion exchange and biliary bicarbonate excretion.
      • Banales J.M.
      • Arenas F.
      • Rodriguez-Ortigosa C.M.
      • Saez E.
      • Uriarte I.
      • Doctor R.B.
      • et al.
      Bicarbonate-rich choleresis induced by secretin in normal rat is taurocholate-dependent and involves AE2 anion exchanger.
      • Alvaro D.
      • Mennone A.
      • Boyer J.L.
      Role of kinases and phosphatases in the regulation of fluid secretion and Cl−/HCO3− exchange in cholangiocytes.
      • Stewart A.K.
      • Chernova M.N.
      • Kunes Y.Z.
      • Alper S.L.
      Regulation of AE2 anion exchanger by intracellular pH: critical regions of the NH(2)-terminal cytoplasmic domain.
      • Cohn J.A.
      • Strong T.V.
      • Picciotto M.R.
      • Nairn A.C.
      • Collins F.S.
      • Fitz J.G.
      Localization of the cystic fibrosis transmembrane conductance regulator in human bile duct epithelial cells.
      • Fitz J.G.
      • Basavappa S.
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      • Melhus O.
      • Cohn J.A.
      Regulation of membrane chloride currents in rat bile duct epithelial cells.
      Biliary HCO3 secretion induces the alkalinisation and fluidisation of bile and creates a “biliary bicarbonate umbrella” that protects cholangiocytes against the damaging effect of the cytotoxic bile acids secreted through the canalicular BSEP, reducing their protonation status.
      • Banales J.M.
      • Arenas F.
      • Rodriguez-Ortigosa C.M.
      • Saez E.
      • Uriarte I.
      • Doctor R.B.
      • et al.
      Bicarbonate-rich choleresis induced by secretin in normal rat is taurocholate-dependent and involves AE2 anion exchanger.
      • Beuers U.
      • Hohenester S.
      • de Buy Wenniger L.J.
      • Kremer A.E.
      • Jansen P.L.
      • Elferink R.P.
      The biliary HCO(3)(−) umbrella: a unifying hypothesis on pathogenetic and therapeutic aspects of fibrosing cholangiopathies.
      • Hohenester S.
      • Wenniger L.M.
      • Paulusma C.C.
      • van Vliet S.J.
      • Jefferson D.M.
      • Elferink R.P.
      • et al.
      A biliary HCO3− umbrella constitutes a protective mechanism against bile acid-induced injury in human cholangiocytes.
      On the other hand, acetylcholine increases the cytosolic Ca2+ level through an InsP3R3-dependent mechanism, which activates the apical Cl secretion through TMEM16A and subsequent Cl/HCO3 exchange via AE2.
      • Banales J.M.
      • Prieto J.
      • Medina J.F.
      Cholangiocyte anion exchange and biliary bicarbonate excretion.
      • Dutta A.K.
      • Woo K.
      • Khimji A.K.
      • Kresge C.
      • Feranchak A.P.
      Mechanosensitive Cl− secretion in biliary epithelium mediated through TMEM16A.
      • Trampert D.C.
      • Nathanson M.H.
      Regulation of bile secretion by calcium signaling in health and disease.
      In PBC, miR-506 is found upregulated in cholangiocytes where it directly targets both AE2 and InsP3R3, resulting in intracellular pH and Ca2+ alterations and impaired biliary bicarbonate secretion that ends in cholestasis.
      • Banales J.M.
      • Saez E.
      • Uriz M.
      • Sarvide S.
      • Urribarri A.D.
      • Splinter P.
      • et al.
      Up-regulation of microRNA 506 leads to decreased Cl−/HCO3− anion exchanger 2 expression in biliary epithelium of patients with primary biliary cirrhosis.
      • Ananthanarayanan M.
      • Banales J.M.
      • Guerra M.T.
      • Spirli C.
      • Munoz-Garrido P.
      • Mitchell-Richards K.
      • et al.
      Post-translational regulation of the type III inositol 1,4,5-trisphosphate receptor by miRNA-506.
      As a consequence of the decreased biliary bicarbonate secretion, toxic bile acids are able to enter cholangiocytes in an uncontrolled fashion and promote apoptosis. Under these conditions, the mitochondrial energetic metabolism is severely disturbed, ending in decreased ATP production and PDC-E2 overexpression and aberrant localisation, contributing to immune activation.
      • Erice O.
      • Munoz-Garrido P.
      • Vaquero J.
      • Perugorria M.J.
      • Fernandez-Barrena M.G.
      • Saez E.
      • et al.
      MicroRNA-506 promotes primary biliary cholangitis-like features in cholangiocytes and immune activation.
      Ach, acetylcholine; AE2, Na+-independent Cl/HCO3 exchanger 2; AQP1, aquaporin 1; BSEP, bile salt export pump; cAMP, cyclic adenosine monophosphate; CFTR, cystic fibrosis transmembrane conductance regulator; ER, endoplasmic reticulum; FXR, farnesoid X receptor; InsP3R3, type III inositol 1,4,5-trisphosphate receptor; PDC-E2, E2 component of the pyruvate dehydrogenase complex; pHi, intracellular pH; TMEM16A, transmembrane member 16A.
      Future studies should aim to determine the molecular mechanisms that promote miR-506 overexpression in PBC cholangiocytes, and if any of the potential aetiological factors proposed in PBC (environmental, genetic or other epigenetics) regulate its expression. Moreover, the fact that miR-506 is located on the X chromosome and that PBC mainly affects women emphasises the role of the X chromosomes in PBC. Of note, it is not yet clear if AE2 promoter methylation disturbances also occur in patients with PBC, which could account for another epigenetic phenomenon regulating the secretory phenotype, in parallel with miR-506. Altogether, the epigenetic era has opened a new field of research in PBC and an opportunity to deepen our understanding of this complex disease.

      Financial support

      Spanish Ministry of Economy and Competitiveness [J.M. Banales ( PI12/00380 , PI15/01132 , PI18/01075 and Miguel Servet Programme CON14/00129 ), M.J. Perugorria ( Fondo de Investigación en Salud (FIS) PI14/00399 , PI17/00022 and “Ramon y Cajal” Programme RYC-2015-17755 ), cofinanced by “Fondo Europeo de Desarrollo Regional” ( FEDER )]; ISCIII CIBERehd : J.M. Banales, M.J. Perugorria, and L. Bujanda, Spain; “Diputación Foral de Gipuzkoa” (J.M. Banales: DFG15/010 , DFG16/004 ), BIOEF ( Basque Foundation for Innovation and Health Research : EiTB Maratoia BIO15/CA/016/BD to J.M. Banales), Department of Health of the Basque Country (M.J. Perugorria: 2015111100 ; J.M. Banales: 2017111010 ), and “Fundación Científica de la Asociación Española Contra el Cancer” ( AECC Scientific Foundation , to J.M. Banales). A. Santos-Laso was funded by the Basque Government ( PRE_2015_1_0126 ).

      Conflict of interest

      The authors declare no conflicts of interest that pertain to this work.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Supplementary data

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