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Autophagy and senescence in fibrosing cholangiopathies

Open AccessPublished:November 27, 2014DOI:https://doi.org/10.1016/j.jhep.2014.11.027

      Summary

      Fibrosing cholangiopathy such as primary sclerosing cholangitis (PSC) and biliary atresia (BA) is characterized by biliary epithelial injuries and concentric fibrous obliteration of the biliary tree together with inflammatory cell infiltration. In these diseases, inappropriate innate immunity is reported to contribute more to bile duct pathology as compared with various aspects of “classical” autoimmune diseases. Primary biliary cirrhosis (PBC) is characterized by chronic cholangitis with bile duct loss and classical autoimmune features. Cellular senescence of cholangiocytes and a senescence-associated secretory phenotype lead to the production of proinflammatory cytokines and chemokines that may modify the milieu of the bile duct and then trigger fibroinflammatory responses in PSC and PBC. Furthermore, deregulated autophagy might be involved in cholangiocyte senescence and possibly in the autoimmune process in PBC, and the deregulated innate immunity against enteric microbes or their products that is associated with cholangiocyte senescence might result in the fibrosing cholangitis that develops in PBC and PSC. In BA, innate immunity against double-stranded RNA viruses might be involved in cholangiocyte apoptosis and also in the development of the epithelial-mesenchymal transition of cholangiocytes that results in fibrous obliteration of bile ducts. These recent advances in the understanding of immune-mediated biliary diseases represent a paradigm shift: the cholangiocyte is no longer viewed merely as a passive victim of injury; it is now also considered to function as a potential effector in bile duct pathology.

      Abbreviations:

      BA (biliary atresia), EMT (epithelial-mesenchymal transition), PAMP (pathogen-associated molecular patterns), PBC (primary biliary cirrhosis), PSC (primary sclerosing cholangitis), SASP (senescence-associated secretory phenotype), TLR (Toll-like receptor), TRAIL (tumor factor-related apoptosis-inducing ligand), CNSDC (chronic nonsuppurative destructive cholangitis), AMA (antimitochondrial antibodies), SA-β-gal (senescence-associated β-galactosidase), LPS (lipopolysaccharide), MyD88 (myeloid differentiation factor 88), IRAK-1, IL-1 (receptor-associated kinase-1), dsRNA (double-stranded RNA), IRF-3 (interferon regulatory factor 3), PDC-E2 (pyruvate dehydrogenase complex-E2), LC3 (light chain 3β), MHC (major histocompatibility complex), pANCA (perinuclear anti-neutrophil cytoplasmic antibody), BEC-Abs (biliary epithelial cell antibodies)

      Keywords

      Introduction

      The biliary tree is composed of a ramifying tubular network that modifies the composition of bile and serves as a conduit for the delivery of bile to the duodenum [
      • Lazaridis K.N.
      • Strazzabosco M.
      • Larusso N.F.
      The cholangiopathies: disorders of biliary epithelia.
      ,
      • Nakanuma Y.
      • Zen Y.
      • Portman B.C.
      Diseases of the bile duct.
      ,
      • Nakanuma Y.
      • Harada K.
      • Sasaki M.
      • Sato Y.
      Proposal of a new disease concept “biliary diseases with pancreatic counterparts”. Anatomical and pathological bases.
      ,
      • LaRusso N.F.
      • Shneider B.L.
      • Black D.
      • Gores G.J.
      • James S.P.
      • Doo E.
      • et al.
      Primary sclerosing cholangitis: summary of a workshop.
      ]. The epithelial lining cells (cholangiocytes) exhibit heterogeneities in their morphologies, phenotypes and gene expression along the biliary tree and function as a secretory/absorptive epithelium and other physiological roles [
      • O’Hara S.P.
      • Tabibian J.H.
      • Splinter P.L.
      • LaRusso N.F.
      The dynamic biliary epithelia: molecules, pathways, and disease.
      ,
      • Marzioni M.
      • Glaser S.S.
      • Francis H.
      • Phinizy J.L.
      • LeSage G.
      • Alpini G.
      Functional heterogeneity of cholangiocytes.
      ,
      • Kullak-Ublick G.A.
      • Beuers U.
      • Paumgartner G.
      Hepatobiliary transport.
      ,
      • Harada K.
      • Nakanuma Y.
      Cholangiopathy with respect to biliary innate immunity.
      ]. Cholangiopathies are a diverse group of biliary disorders of distinct etiologies, and they include, for example, immune-mediated, drug-induced and infectious cholangiopathies [
      • Lazaridis K.N.
      • Strazzabosco M.
      • Larusso N.F.
      The cholangiopathies: disorders of biliary epithelia.
      ,
      • Nakanuma Y.
      • Zen Y.
      • Portman B.C.
      Diseases of the bile duct.
      ,
      • Tabibian J.H.
      • O’Hara S.P.
      • Lindor K.D.
      Primary sclerosing cholangitis and the microbiota: current knowledge and perspectives on etiopathogenesis and emerging therapies.
      ,
      • Aron J.H.
      • Bowlus C.L.
      The immunobiology of primary sclerosing cholangitis.
      ,
      • Nakanuma Y.
      • Harada K.
      • Sato Y.
      • Ikeda H.
      Recent progress in the etiopathogenesis of pediatric biliary disease, particularly Caroli’s disease with congenital hepatic fibrosis and biliary atresia.
      ,
      • Nakanuma Y.
      • Harada K.
      • Katayanagi K.
      • Tsuneyama K.
      • Sasaki M.
      Definition and pathology of primary sclerosing cholangitis.
      ]. Among them, fibrosing cholangiopathies such as primary sclerosing cholangitis (PSC) and biliary atresia (BA) are characterized by cholangiocytic injuries and progressive fibrous obliteration of the biliary tree associated with nonspecific inflammatory cell infiltration [
      • Lazaridis K.N.
      • Strazzabosco M.
      • Larusso N.F.
      The cholangiopathies: disorders of biliary epithelia.
      ,
      • Nakanuma Y.
      • Zen Y.
      • Portman B.C.
      Diseases of the bile duct.
      ,
      • Tabibian J.H.
      • O’Hara S.P.
      • Lindor K.D.
      Primary sclerosing cholangitis and the microbiota: current knowledge and perspectives on etiopathogenesis and emerging therapies.
      ,
      • Aron J.H.
      • Bowlus C.L.
      The immunobiology of primary sclerosing cholangitis.
      ,
      • Nakanuma Y.
      • Harada K.
      • Sato Y.
      • Ikeda H.
      Recent progress in the etiopathogenesis of pediatric biliary disease, particularly Caroli’s disease with congenital hepatic fibrosis and biliary atresia.
      ,
      • Nakanuma Y.
      • Harada K.
      • Katayanagi K.
      • Tsuneyama K.
      • Sasaki M.
      Definition and pathology of primary sclerosing cholangitis.
      ]. Whereas PSC and BA are considered complex disorders involving multiple etiopathogenetic factors, immune system-mediated assaults against the cholangiocytes are regarded a central feature [
      • Lazaridis K.N.
      • Strazzabosco M.
      • Larusso N.F.
      The cholangiopathies: disorders of biliary epithelia.
      ,
      • Nakanuma Y.
      • Zen Y.
      • Portman B.C.
      Diseases of the bile duct.
      ,
      • Tabibian J.H.
      • O’Hara S.P.
      • Lindor K.D.
      Primary sclerosing cholangitis and the microbiota: current knowledge and perspectives on etiopathogenesis and emerging therapies.
      ,
      • Aron J.H.
      • Bowlus C.L.
      The immunobiology of primary sclerosing cholangitis.
      ,
      • Nakanuma Y.
      • Harada K.
      • Sato Y.
      • Ikeda H.
      Recent progress in the etiopathogenesis of pediatric biliary disease, particularly Caroli’s disease with congenital hepatic fibrosis and biliary atresia.
      ,
      • Nakanuma Y.
      • Harada K.
      • Katayanagi K.
      • Tsuneyama K.
      • Sasaki M.
      Definition and pathology of primary sclerosing cholangitis.
      ,
      • Harada K.
      • Sato Y.
      • Itatsu K.
      • Isse K.
      • Ikeda H.
      • Yasoshima M.
      • et al.
      Innate immune response to double-stranded RNA in biliary epithelial cells is associated with the pathogenesis of biliary atresia.
      ,
      • Harada K.
      • Sato Y.
      • Ikeda H.
      • Isse K.
      • Ozaki S.
      • Enomae M.
      • et al.
      Epithelial-mesenchymal transition induced by biliary innate immunity contributes to the sclerosing cholangiopathy of biliary atresia.
      ,
      • Szavay P.O.
      • Leonhardt J.
      • Czech-Schmidt G.
      • Petersen C.
      The role of reovirus type 3 infection in an established murine model for biliary atresia.
      ,
      • Feldman A.G.
      • Mack C.L.
      Biliary atresia: cellular dynamics and immune dysregulation.
      ]. Primary biliary cirrhosis (PBC), another form of fibrosing cholangiopathy [
      • 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.
      ,
      • EASL Clinical Practice Guidelines
      Management of cholestatic liver diseases.
      ], is associated with marked lymphoplasmacytic infiltration around the interlobular ducts (chronic nonsuppurative destructive cholangitis (CNSDC)) and classical autoimmune features including disease specific antimitochondrial antibodies (AMAs) [
      • Hirschfield G.M.
      • Gershwin M.E.
      The immunobiology and pathophysiology of primary biliary cirrhosis.
      ].
      In response to diverse stresses, cholangiocytes exhibit various passive and adverse cytopathic changes such as cellular swelling or shrinkage. This is followed either by the repair or regeneration of cholangiocytes or by bile duct loss through apoptosis or necrosis [
      • Nakanuma Y.
      • Zen Y.
      • Portman B.C.
      Diseases of the bile duct.
      ,
      • Harada K.
      • Ozaki S.
      • Gershwin M.E.
      • Nakanuma Y.
      Enhanced apoptosis relates to bile duct loss in primary biliary cirrhosis.
      ]; the pathology and pathogenesis of these processes have been extensively studied [
      • Nakanuma Y.
      • Zen Y.
      • Portman B.C.
      Diseases of the bile duct.
      ,
      • Harada K.
      • Nakanuma Y.
      Cholangiopathy with respect to biliary innate immunity.
      ,
      • Harada K.
      • Ozaki S.
      • Gershwin M.E.
      • Nakanuma Y.
      Enhanced apoptosis relates to bile duct loss in primary biliary cirrhosis.
      ,
      • Sasaki M.
      • Nakanuma Y.
      Biliary epithelial apoptosis, autophagy, and senescence in primary biliary cirrhosis.
      ]. However, recent studies have engendered a novel paradigm of the biliary epithelial pathophysiology: cholangiocytes, which were once considered to act as a passive victim under pathologic conditions, are now recognized as active players in the biliary proinflammatory response to various stresses [
      • Harada K.
      • Nakanuma Y.
      Cholangiopathy with respect to biliary innate immunity.
      ,
      • Harada K.
      • Sato Y.
      • Ikeda H.
      • Isse K.
      • Ozaki S.
      • Enomae M.
      • et al.
      Epithelial-mesenchymal transition induced by biliary innate immunity contributes to the sclerosing cholangiopathy of biliary atresia.
      ,
      • Sasaki M.
      • Nakanuma Y.
      Biliary epithelial apoptosis, autophagy, and senescence in primary biliary cirrhosis.
      ,
      • Harada K.
      • Isse K.
      • Kamihira T.
      • Shimoda S.
      • Nakanuma Y.
      Th1 cytokine-induced downregulation of PPARγ in human biliary cells relates to cholangitis in primary biliary cirrhosis.
      ,
      • Yasoshima M.
      • Tsuneyama K.
      • Harada K.
      • Sasaki M.
      • Gershwin M.E.
      • Nakanuma Y.
      Immunohistochemical analysis of cell-matrix adhesion molecules and their ligands in the portal tracts of primary biliary cirrhosis.
      ].
      Figure thumbnail fx1

      Cellular senescence and autophagy with respect to apoptosis

      Cells respond to various stresses by means of adaptation, autophagy, and recovery, or they either commit to irreversible cell-cycle exit (senescence) or are eliminated through programmed cell death (apoptosis) [
      • White E.
      • Low S.W.
      Eating to exit: autophagy-enabled senescence revealed.
      ]. While cellular senescence, autophagy and apoptosis are distinct cellular responses to stress, they are correlating with each other, and signaling pathways involved are often overlapping each other [
      • Collado M.
      • Blasco M.A.
      • Serrano M.
      Cellular senescence in cancer and aging.
      ,
      • Iwasa H.
      • Han J.
      • Ishikawa F.
      Mitogen-activated protein kinase p38 defines the common senescence-signalling pathway.
      ,
      • Gitenay D.
      • Lallet-Daher H.
      • Bernard D.
      Caspase-2 regulates oncogene-induced senescence.
      ,
      • de Graaf E.L.
      • Kaplon J.
      • Zhou H.
      • Heck A.J.
      • Peeper D.S.
      • Altelaar A.F.
      Phosphoproteome dynamics in onset and maintenance of oncogene-induced senescence.
      ,
      • Zhu B.
      • Ferry C.H.
      • Markell L.K.
      • Blazanin N.
      • Glick A.B.
      • Gonzalez F.J.
      • et al.
      The nuclear receptor peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) promotes oncogene-induced cellular senescence through repression of endoplasmic reticulum stress.
      ,
      • Strozyk E.
      • Kulms D.
      The role of AKT/mTOR pathway in stress response to UV-irradiation: implication in skin carcinogenesis by regulation of apoptosis, autophagy and senescence.
      ,
      • Kalimuthu S.
      • Se-Kwon K.
      Cell survival and apoptosis signaling as therapeutic target for cancer: marine bioactive compounds.
      ,
      • Lockshin R.A.
      • Zakeri Z.
      Cell death in health and disease.
      ]. For example, autophagy is considered a survival mechanism when faced with cellular insults, however extensive autophagy can also lead to senescence or apoptosis (Fig. 1) [
      • Strozyk E.
      • Kulms D.
      The role of AKT/mTOR pathway in stress response to UV-irradiation: implication in skin carcinogenesis by regulation of apoptosis, autophagy and senescence.
      ,
      • Kalimuthu S.
      • Se-Kwon K.
      Cell survival and apoptosis signaling as therapeutic target for cancer: marine bioactive compounds.
      ,
      • Lockshin R.A.
      • Zakeri Z.
      Cell death in health and disease.
      ,
      • Young A.R.
      • Narita M.
      • Ferreira M.
      • Kirschner K.
      • Sadaie M.
      • Darot J.F.
      • et al.
      Autophagy mediates the mitotic senescence transition.
      ,
      • Sasaki M.
      • Ikeda H.
      • Nakanuma Y.
      Activation of ATM signaling pathway is involved in oxidative stress-induced expression of mito-inhibitory p21WAF1/Cip1 in chronic non- suppurative destructive cholangitis in primary biliary cirrhosis: an immunohistochemical study.
      ,
      • Collado M.
      • Blasco M.A.
      • Serrano M.
      Cellular senescence in cancer and aging.
      ,
      • Harper J.W.
      • Elledge S.J.
      The DNA damage response: ten years after.
      ].
      Figure thumbnail gr1
      Fig. 1Cellular responses to stress. Apoptosis, autophagy, and cellular senescence are distinct cellular responses to stress. Autophagy is induced in response to a wide variety of stresses, and there are many purposes for autophagy such as the quality control of protein and organelle, damage mitigation, and energy homeostasis. Stressed cells activate autophagy, which prevents damage and maintains metabolism though lysosomal turnover of cellular components. Autophagy can facilitate senescence or limit damage and delay apoptosis to allow recovery and repair of normal cell function (modified from
      [
      • Nakanuma Y.
      • Harada K.
      • Katayanagi K.
      • Tsuneyama K.
      • Sasaki M.
      Definition and pathology of primary sclerosing cholangitis.
      ]
      ).

      Cellular senescence

      Senescence is a condition in which a cell can no longer proliferate and senescent cells are irreversibly arrested at the G1 phase [
      • Collado M.
      • Blasco M.A.
      • Serrano M.
      Cellular senescence in cancer and aging.
      ,
      • Gitenay D.
      • Lallet-Daher H.
      • Bernard D.
      Caspase-2 regulates oncogene-induced senescence.
      ,
      • Kuilman T.
      • Michaloglou C.
      • Mooi W.J.
      • Peeper D.S.
      The essence of senescence.
      ]. These cells do not respond to various external stimuli, but they remain metabolically active. Senescence can be triggered by multiple mechanisms, including derepression of the INK4a/ARF locus, telomere dysfunction, oxidative stress, and oncogene activation [
      • Collado M.
      • Blasco M.A.
      • Serrano M.
      Cellular senescence in cancer and aging.
      ,
      • Iwasa H.
      • Han J.
      • Ishikawa F.
      Mitogen-activated protein kinase p38 defines the common senescence-signalling pathway.
      ,
      • Gitenay D.
      • Lallet-Daher H.
      • Bernard D.
      Caspase-2 regulates oncogene-induced senescence.
      ,
      • de Graaf E.L.
      • Kaplon J.
      • Zhou H.
      • Heck A.J.
      • Peeper D.S.
      • Altelaar A.F.
      Phosphoproteome dynamics in onset and maintenance of oncogene-induced senescence.
      ,
      • d’Adda di Fagagna F.
      • Reaper P.M.
      • Clay-Farrace L.
      • Fiegler H.
      • Carr P.
      • Von Zglinicki T.
      • et al.
      A DNA damage checkpoint response in telomere-initiated senescence.
      ]. In contrast to apoptosis, senescence is typically a delayed stress response that involves multiple effector mechanisms, and it can serve a program that protects against cellular insults [
      • Lawless C.
      • Wang C.
      • Jurk D.
      • Merz A.
      • Zglinicki T.V.
      • Passos J.F.
      Quantitative assessment of markers for cell senescence.
      ,
      • Levine B.
      • Kroemer G.
      Autophagy in the pathogenesis of disease.
      ]. Senescence is regarded as a mechanism responsible for irreversibly blocking the proliferation of cells that harbor genomic injuries [
      • Collado M.
      • Blasco M.A.
      • Serrano M.
      Cellular senescence in cancer and aging.
      ,
      • Gitenay D.
      • Lallet-Daher H.
      • Bernard D.
      Caspase-2 regulates oncogene-induced senescence.
      ]. Senescent cells display several distinctive characteristics such as unique histological changes [
      • Sigal S.H.
      • Rajvanshi P.
      • Gorla G.R.
      • Sokhi R.P.
      • Saxena R.
      • Gebhard Jr., D.R.
      • et al.
      Partial hepatectomy-induced polyploidy attenuates hepatocyte replication and activates cell aging events.
      ,
      • Brodsky W.Y.
      • Uryvaeva I.V.
      Cell polyploidy: its relation to tissue growth and function.
      ], increased expression of p16INK4 and p21WAF1/Cip, and increased activity of senescence-associated β-galactosidase (SA-β-gal) [
      • Dimri G.P.
      • Lee X.
      • Basile G.
      • Acosta M.
      • Scott G.
      • Roskelley C.
      • et al.
      A biomarker that identifies senescent human cells in culture and in aging skin in vivo.
      ]. As well as progression to a senescence-associated secretory phenotype (SASP), which is a persistent hypersecretory state characterized by increased expression and secretion of inflammatory molecules such as cytokines, chemokines, growth factors and profibrotic factors [
      • O’Hara S.P.
      • Tabibian J.H.
      • Splinter P.L.
      • LaRusso N.F.
      The dynamic biliary epithelia: molecules, pathways, and disease.
      ,
      • Kuilman T.
      • Michaloglou C.
      • Mooi W.J.
      • Peeper D.S.
      The essence of senescence.
      ,
      • Lawless C.
      • Wang C.
      • Jurk D.
      • Merz A.
      • Zglinicki T.V.
      • Passos J.F.
      Quantitative assessment of markers for cell senescence.
      ,
      • Sasaki M.
      • Ikeda H.
      • Haga H.
      • Manabe T.
      • Nakanuma Y.
      Frequent cellular senescence in small bile ducts in primary biliary cirrhosis: a possible role in bile duct loss.
      ,
      • Sasaki M.
      • Miyakoshi M.
      • Sato Y.
      • Nakanuma Y.
      Modulation of the microenvironment by senescent biliary epithelial cells may be involved in the pathogenesis of primary biliary cirrhosis.
      ,
      • Coppé J.P.
      • Patil C.K.
      • Rodier F.
      • Sun Y.
      • Muñoz D.P.
      • Goldstein J.
      • et al.
      Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor.
      ,
      • Wajapeyee N.
      • Serra R.W.
      • Zhu X.
      • Mahalingam M.
      • Green M.R.
      Oncogenic BRAF induces senescence and apoptosis through pathways mediated by the secreted protein IGFBP7.
      ,
      • Tabibian J.H.
      • Trussoni C.E.
      • O’Hara S.P.
      • Splinter P.L.
      • Heimbach J.K.
      • LaRusso N.F.
      Characterization of cultured cholangiocytes isolated from livers of patients with primary sclerosing cholangitis.
      ,
      • Tabibian J.H.
      • O’Hara S.P.
      • Splinter P.L.
      • Trussoni C.E.
      • LaRusso N.F.
      Cholangiocyte senescence by way of N-ras activation is a characteristic of primary sclerosing cholangitis.
      ,
      • Acosta J.C.
      • O’Loghlen A.
      • Banito A.
      • Guijarro M.V.
      • Augert A.
      • Raguz S.
      • et al.
      Chemokine signaling via the CXCR2 receptor reinforces senescence.
      ,
      • Kuilman T.
      • Michaloglou C.
      • Vredeveld L.C.
      • Douma S.
      • van Doorn R.
      • Desmet C.J.
      • et al.
      Oncogene-induced senescence relayed by an interleukin-dependent inflammatory network.
      ].

      Autophagy

      Autophagy is a genetically regulated program for self-degradation aimed to remove long-lived or damaged proteins and organelles, and for recycling of cytoplasmic content [
      • Levine B.
      • Kroemer G.
      Autophagy in the pathogenesis of disease.
      ,
      • Mizushima N.
      Autophagy: process and function.
      ,
      • Yin X.M.
      • Ding W.X.
      • Gao W.
      Autophagy in the liver.
      ]. Autophagy maintains cellular viability during periods of various stresses [
      • Strozyk E.
      • Kulms D.
      The role of AKT/mTOR pathway in stress response to UV-irradiation: implication in skin carcinogenesis by regulation of apoptosis, autophagy and senescence.
      ,
      • Kalimuthu S.
      • Se-Kwon K.
      Cell survival and apoptosis signaling as therapeutic target for cancer: marine bioactive compounds.
      ,
      • Lockshin R.A.
      • Zakeri Z.
      Cell death in health and disease.
      ,
      • Levine B.
      • Kroemer G.
      Autophagy in the pathogenesis of disease.
      ,
      • Mizushima N.
      Autophagy: process and function.
      ,
      • Yin X.M.
      • Ding W.X.
      • Gao W.
      Autophagy in the liver.
      ]. During cellular stress, autophagy is activated and organelles are sequestered in autophagosomes and digested through fusion with lysosomes. Autophagy can act both as a temporary protective mechanism during a brief stressful episode and the adaptive response of autophagy enables the cells to maintain homeostasis and to survive starvation stress [
      • Kroemer G.
      • Mariño G.
      • Levine B.
      Autophagy and the integrated stress response.
      ]. However, depending on the stress or stimuli autophagy also contributes to cell death, called autophagic cell death [
      • Amelio I.
      • Melino G.
      • Knight R.A.
      Cell death pathology: cross-talk with autophagy and its clinical implications.
      ].

      Relations among apoptosis, autophagy and cellular senescence

      Recently, autophagy has been identified as an effector mechanism of senescence [
      • Young A.R.
      • Narita M.
      • Ferreira M.
      • Kirschner K.
      • Sadaie M.
      • Darot J.F.
      • et al.
      Autophagy mediates the mitotic senescence transition.
      ]. During senescence, autophagy is induced and activated, consequently facilitating the process of senescence. Furthermore, inhibition of autophagy delayed the senescence phenotype [
      • Young A.R.
      • Narita M.
      • Ferreira M.
      • Kirschner K.
      • Sadaie M.
      • Darot J.F.
      • et al.
      Autophagy mediates the mitotic senescence transition.
      ]. Autophagy is regulated by several kinases, particularly mTOR and AKT [
      • Strozyk E.
      • Kulms D.
      The role of AKT/mTOR pathway in stress response to UV-irradiation: implication in skin carcinogenesis by regulation of apoptosis, autophagy and senescence.
      ,
      • Kalimuthu S.
      • Se-Kwon K.
      Cell survival and apoptosis signaling as therapeutic target for cancer: marine bioactive compounds.
      ]. Apoptosis is central to various physiological processes and the maintenance of homeostasis [
      • Lockshin R.A.
      • Zakeri Z.
      Cell death in health and disease.
      ], its induction eliminates damaged cells mediated through the action of tumor suppressor p53 [
      • Lockshin R.A.
      • Zakeri Z.
      Cell death in health and disease.
      ]. However, in order to prevent excessive loss of injured cells, apoptotic pathways are counteracted by anti-apoptotic signaling including the AKT/mTOR pathway [
      • Strozyk E.
      • Kulms D.
      The role of AKT/mTOR pathway in stress response to UV-irradiation: implication in skin carcinogenesis by regulation of apoptosis, autophagy and senescence.
      ,
      • Kalimuthu S.
      • Se-Kwon K.
      Cell survival and apoptosis signaling as therapeutic target for cancer: marine bioactive compounds.
      ,
      • Lockshin R.A.
      • Zakeri Z.
      Cell death in health and disease.
      ]. AKT/mTOR not only prevents cell death, but is active in cell cycle transition, thereby also counteracting p53. The balance between p53 and AKT/mTOR determines the fate of injured cells. AKT/mTOR is tuned down by the negative regulators being controlled by the p53. This inhibition of AKT/mTOR, in combination with transactivation of damage-regulated autophagy modulators, guides the p53-mediated elimination of damaged cellular components by autophagic clearance. Alternatively, p53 irreversibly blocks cell cycle progression to prevent AKT/mTOR-driven proliferation, thereby inducing premature senescence [
      • Strozyk E.
      • Kulms D.
      The role of AKT/mTOR pathway in stress response to UV-irradiation: implication in skin carcinogenesis by regulation of apoptosis, autophagy and senescence.
      ,
      • Kalimuthu S.
      • Se-Kwon K.
      Cell survival and apoptosis signaling as therapeutic target for cancer: marine bioactive compounds.
      ,
      • Lockshin R.A.
      • Zakeri Z.
      Cell death in health and disease.
      ]. Under slightly different physiological conditions, concomitant activation of p53 and AKT/mTOR can drive cells into senescence. Cross talk and interaction between AKT/mTOR and p53 influences the cell response deciding over death or survival [
      • Strozyk E.
      • Kulms D.
      The role of AKT/mTOR pathway in stress response to UV-irradiation: implication in skin carcinogenesis by regulation of apoptosis, autophagy and senescence.
      ,
      • Kalimuthu S.
      • Se-Kwon K.
      Cell survival and apoptosis signaling as therapeutic target for cancer: marine bioactive compounds.
      ,
      • Lockshin R.A.
      • Zakeri Z.
      Cell death in health and disease.
      ,
      • Amelio I.
      • Melino G.
      • Knight R.A.
      Cell death pathology: cross-talk with autophagy and its clinical implications.
      ,
      • Hiramatsu K.
      • Harada K.
      • Tsuneyama K.
      • Sasaki M.
      • Fujita S.
      • Hashimoto T.
      • et al.
      Amplification and sequence analysis of partial bacterial 16S ribosomal RNA gene in gallbladder bile from patients with primary biliary cirrhosis.
      ].

      Significance of cholangiocyte senescence with respect to deregulated innate immunity

      Cholangiocytes are continuously exposed to bile: in addition to endogenous bile constituents, cholangiocytes can be consistently exposed to microbes or their products such as lipopolysaccharide (LPS) that are cleared from portal venous blood by hepatocytes and are secreted into bile under normal and pathologic conditions [
      • Nakanuma Y.
      • Zen Y.
      • Portman B.C.
      Diseases of the bile duct.
      ,
      • LaRusso N.F.
      • Shneider B.L.
      • Black D.
      • Gores G.J.
      • James S.P.
      • Doo E.
      • et al.
      Primary sclerosing cholangitis: summary of a workshop.
      ,
      • Hiramatsu K.
      • Harada K.
      • Tsuneyama K.
      • Sasaki M.
      • Fujita S.
      • Hashimoto T.
      • et al.
      Amplification and sequence analysis of partial bacterial 16S ribosomal RNA gene in gallbladder bile from patients with primary biliary cirrhosis.
      ]. These substances potentially elicit proinflammatory and chemotactic responses. However, cholangiocytes in healthy livers are tightly regulated in order to prevent the development of abnormal responses to such endogenous or exogenous bile components [
      • Hiramatsu K.
      • Harada K.
      • Tsuneyama K.
      • Sasaki M.
      • Fujita S.
      • Hashimoto T.
      • et al.
      Amplification and sequence analysis of partial bacterial 16S ribosomal RNA gene in gallbladder bile from patients with primary biliary cirrhosis.
      ].
      Recently, cholangiocytes have been widely reported to function as active players in the bile duct pathologies in several cholangiopathies, including PBC and PSC [
      • Harada K.
      • Nakanuma Y.
      Cholangiopathy with respect to biliary innate immunity.
      ,
      • Harada K.
      • Sato Y.
      • Itatsu K.
      • Isse K.
      • Ikeda H.
      • Yasoshima M.
      • et al.
      Innate immune response to double-stranded RNA in biliary epithelial cells is associated with the pathogenesis of biliary atresia.
      ,
      • Sasaki M.
      • Nakanuma Y.
      Biliary epithelial apoptosis, autophagy, and senescence in primary biliary cirrhosis.
      ,
      • Harada K.
      • Isse K.
      • Kamihira T.
      • Shimoda S.
      • Nakanuma Y.
      Th1 cytokine-induced downregulation of PPARγ in human biliary cells relates to cholangitis in primary biliary cirrhosis.
      ,
      • Isse K.
      • Harada K.
      • Zen Y.
      • Kamihira T.
      • Shimoda S.
      • Harada M.
      • et al.
      Fractalkine and CX3CR1 are involved in the recruitment of intraepithelial lymphocytes of intrahepatic bile ducts.
      ,
      • Harada K.
      • Isse K.
      • Sato Y.
      • Ozaki S.
      • Nakanuma Y.
      Endotoxin tolerance in human intrahepatic biliary epithelial cells is induced by upregulation of IRAK-M.
      ,
      • Harada K.
      • Shimoda S.
      • Ikeda H.
      • Chiba M.
      • Hsu M.
      • Sato Y.
      • et al.
      Significance of periductal Langerhans cells and biliary epithelial cell-derived macrophage inflammatory protein-3α in the pathogenesis of primary biliary cirrhosis.
      ,
      • Shimoda S.
      • Harada K.
      • Niiro H.
      • Shirabe K.
      • Taketomi A.
      • Maehara Y.
      • et al.
      Interaction between Toll-like receptors and natural killer cells in the destruction of bile ducts in primary biliary cirrhosis.
      ,
      • Harada K.
      • Chiba M.
      • Okamura A.
      • Hsu M.
      • Sato Y.
      • Igarashi S.
      • et al.
      Monocyte chemoattractant protein-1 derived from biliary innate immunity contributes to hepatic fibrogenesis.
      ,
      • Ikeda H.
      • Sasaki M.
      • Ishikawa A.
      • Sato Y.
      • Harada K.
      • Zen Y.
      • et al.
      Interaction of Toll-like receptors with bacterial components induces expression of CDX2 and MUC2 in rat biliary epithelium in vivo and in culture.
      ,
      • Sasaki M.
      • Ikeda H.
      • Sato Y.
      • Nakanuma Y.
      Proinflammatory cytokine-induced cellular senescence of biliary epithelial cells is mediated via oxidative stress and activation of ATM pathway: a culture study.
      ,
      • Mizushima N.
      • Levine B.
      • Klionsky D.J.
      Autophagy fights disease through cellular self-digestion.
      ]. The cholangiocytes can acquire an activated phenotype and aberrantly express various receptors, chemokines, cytokines, and other growth factors, they can also interact with pathogen-associated molecular patterns (PAMPs). Specifically, recent studies have shown that the deregulation of innate immunity and the induction of cellular senescence in cholangiocytes are involved in the pathogenesis of immune-mediated biliary diseases [
      • Harada K.
      • Nakanuma Y.
      Cholangiopathy with respect to biliary innate immunity.
      ,
      • Harada K.
      • Sato Y.
      • Itatsu K.
      • Isse K.
      • Ikeda H.
      • Yasoshima M.
      • et al.
      Innate immune response to double-stranded RNA in biliary epithelial cells is associated with the pathogenesis of biliary atresia.
      ,
      • Sasaki M.
      • Nakanuma Y.
      Biliary epithelial apoptosis, autophagy, and senescence in primary biliary cirrhosis.
      ,
      • Harada K.
      • Isse K.
      • Kamihira T.
      • Shimoda S.
      • Nakanuma Y.
      Th1 cytokine-induced downregulation of PPARγ in human biliary cells relates to cholangitis in primary biliary cirrhosis.
      ,
      • Sasaki M.
      • Ikeda H.
      • Haga H.
      • Manabe T.
      • Nakanuma Y.
      Frequent cellular senescence in small bile ducts in primary biliary cirrhosis: a possible role in bile duct loss.
      ,
      • Isse K.
      • Harada K.
      • Zen Y.
      • Kamihira T.
      • Shimoda S.
      • Harada M.
      • et al.
      Fractalkine and CX3CR1 are involved in the recruitment of intraepithelial lymphocytes of intrahepatic bile ducts.
      ]. The activation of the Toll-like receptor (TLR) family upon ligation by PAMPs and the induction of SASP are respectively reported to trigger the secretion of proinflammatory molecules from the cholangiocytes, and these might initiate and modulate bile duct pathology [
      • Sasaki M.
      • Nakanuma Y.
      Biliary epithelial apoptosis, autophagy, and senescence in primary biliary cirrhosis.
      ,
      • Sasaki M.
      • Miyakoshi M.
      • Sato Y.
      • Nakanuma Y.
      Modulation of the microenvironment by senescent biliary epithelial cells may be involved in the pathogenesis of primary biliary cirrhosis.
      ,
      • Sasaki M.
      • Nakanuma Y.
      Novel approach to bile duct damage in primary biliary cirrhosis: participation of cellular senescence and autophagy.
      ].

      Cholangiocyte senescence

      Cholangiocyte senescence is reported to be involved in the immune-mediated bile duct pathology [
      • Lunz III, J.G.
      • Contrucci S.
      • Ruppert K.
      • Ruppert K.
      • Murase N.
      • Fung J.J.
      • et al.
      Replicative senescence of biliary epithelial cells precedes bile duct loss in chronic liver allograft rejection: increased expression of p21WAF1/Cip1 as a disease marker and the influence of immunosuppressive drugs.
      ,
      • Sasaki M.
      • Miyakoshi M.
      • Sato Y.
      • Nakanuma Y.
      Autophagy may precede cellular senescence of bile ductular cells in ductular reaction in primary biliary cirrhosis.
      ,
      • Sasaki M.
      • Miyakoshi M.
      • Sato Y.
      • Nakanuma Y.
      A possible involvement of p62/sequestosome-1 in the process of biliary epithelial autophagy and senescence in primary biliary cirrhosis.
      ]. For example, in chronic liver-allograft rejection which is characterized by a progressive loss of intrahepatic bile ducts [
      • Lunz III, J.G.
      • Contrucci S.
      • Ruppert K.
      • Ruppert K.
      • Murase N.
      • Fung J.J.
      • et al.
      Replicative senescence of biliary epithelial cells precedes bile duct loss in chronic liver allograft rejection: increased expression of p21WAF1/Cip1 as a disease marker and the influence of immunosuppressive drugs.
      ,
      • Brain J.G.
      • Robertson H.
      • Thompson E.
      • Humphreys E.H.
      • Gardner A.
      • Booth T.A.
      • et al.
      Biliary epithelial senescence and plasticity in acute cellular rejection.
      ], the expression of senescence-related p21WAF1/Cip protein is increased in cholangiocytes during early chronic rejection [
      • Lunz III, J.G.
      • Contrucci S.
      • Ruppert K.
      • Ruppert K.
      • Murase N.
      • Fung J.J.
      • et al.
      Replicative senescence of biliary epithelial cells precedes bile duct loss in chronic liver allograft rejection: increased expression of p21WAF1/Cip1 as a disease marker and the influence of immunosuppressive drugs.
      ]. Replicative senescence accounts for the characteristic cytological alterations of cholangiocytes that is used for the diagnosis of early chronic rejection. Unsuccessful recovery from cholangiocyte senescence might be responsible for the progressive bile duct loss in chronic allograft rejection [
      • Lunz III, J.G.
      • Contrucci S.
      • Ruppert K.
      • Ruppert K.
      • Murase N.
      • Fung J.J.
      • et al.
      Replicative senescence of biliary epithelial cells precedes bile duct loss in chronic liver allograft rejection: increased expression of p21WAF1/Cip1 as a disease marker and the influence of immunosuppressive drugs.
      ].

      Cellular senescence and SASP

      Senescent cholangiocytes can play a critical role in modulating the microenvironment by secreting the proinflammatory molecules belonging to SASP. Whereas senescence and SASPs contribute to tissue-repair processes, the persistence of senescent cells and SASPs might have pathologic consequences such as persistent tissue inflammation and impaired tissue integrity, and are reported to be involved in the pathogenesis of diverse human diseases including biliary diseases [
      • Paradis V.
      • Youssef N.
      • Dargère D.
      • Bâ N.
      • Bonvoust F.
      • Deschatrette J.
      • et al.
      Replicative senescence in normal liver, chronic hepatitis C, and hepatocellular carcinomas.
      ,
      • Chilosi M.
      • Carloni A.
      • Rossi A.
      • Poletti V.
      Premature lung aging and cellular senescence in the pathogenesis of idiopathic pulmonary fibrosis and COPD/emphysema.
      ,
      • Kumar M.
      • Seeger W.
      • Voswinckel R.
      Senescence-associated secretory phenotype and its possible role in chronic obstructive pulmonary disease.
      ].

      Bystander effect

      Recent studies [
      • Sasaki M.
      • Miyakoshi M.
      • Sato Y.
      • Nakanuma Y.
      Modulation of the microenvironment by senescent biliary epithelial cells may be involved in the pathogenesis of primary biliary cirrhosis.
      ,
      • Tabibian J.H.
      • O’Hara S.P.
      • Splinter P.L.
      • Trussoni C.E.
      • LaRusso N.F.
      Cholangiocyte senescence by way of N-ras activation is a characteristic of primary sclerosing cholangitis.
      ,
      • Sasaki M.
      • Nakanuma Y.
      Novel approach to bile duct damage in primary biliary cirrhosis: participation of cellular senescence and autophagy.
      ] suggest that cytokines/chemokines produced by senescent cholangiocytes could also facilitate cellular senescence in bystander cholangiocytes and other types of cells in the periductal tissue. For example, we have shown that CCL2 and CX3CL1, which are components of the SASPs of senescent cholangiocytes, induced cellular senescence in cholangiocytes [
      • Sasaki M.
      • Miyakoshi M.
      • Sato Y.
      • Nakanuma Y.
      Modulation of the microenvironment by senescent biliary epithelial cells may be involved in the pathogenesis of primary biliary cirrhosis.
      ,
      • Tabibian J.H.
      • O’Hara S.P.
      • Splinter P.L.
      • Trussoni C.E.
      • LaRusso N.F.
      Cholangiocyte senescence by way of N-ras activation is a characteristic of primary sclerosing cholangitis.
      ,
      • Sasaki M.
      • Nakanuma Y.
      Novel approach to bile duct damage in primary biliary cirrhosis: participation of cellular senescence and autophagy.
      ]. This effect may lead to the accumulation of senescent cholangiocytes in the affected ducts and also senescent mesenchymal cells around the bile ducts in fibrosing cholangiopathies.

      Senescence and phenotypic dedifferentiation of cholangiocytes

      Recently, Brain et al. demonstrated a key role of senescence in acute cellular rejection [
      • Brain J.G.
      • Robertson H.
      • Thompson E.
      • Humphreys E.H.
      • Gardner A.
      • Booth T.A.
      • et al.
      Biliary epithelial senescence and plasticity in acute cellular rejection.
      ]. Acute liver-allograft sections showed a positive correlation between increasing grades of acute rejection and the number of cholangiocytes expressing the senescence marker p21WAF1/Cip or the mesenchymal marker S100A4. Interestingly, in the in vitro study, oxidative stress induced expression of the senescent markers transiently in cultured cholangiocytes. Subsequently, the cholangiocytes exhibited increased expression of S100A4 coupled with a decrease of epithelial markers. This dedifferentiation was associated with production of TGF-β2 by senescent cholangiocytes [
      • Brain J.G.
      • Robertson H.
      • Thompson E.
      • Humphreys E.H.
      • Gardner A.
      • Booth T.A.
      • et al.
      Biliary epithelial senescence and plasticity in acute cellular rejection.
      ]. They suggested that although some of the injured cholangiocytes died in acute allograft rejection, many of them survived in the functionally compromised state of senescence and phenotypic dedifferentiation that was coupled with the acquisition of the mesenchymal marker. This might be related to the epithelial-mesenchymal transition (EMT) of cholangiocytes and progressive fibrosis around the bile duct, and could potentially represent a novel role of senescent cholangiocytes.

      Cellular senescence in relation to innate immunity

      As described above, proinflammatory molecules are secreted by senescent cholangiocytes and SASPs [
      • Kuilman T.
      • Michaloglou C.
      • Mooi W.J.
      • Peeper D.S.
      The essence of senescence.
      ,
      • Wajapeyee N.
      • Serra R.W.
      • Zhu X.
      • Mahalingam M.
      • Green M.R.
      Oncogenic BRAF induces senescence and apoptosis through pathways mediated by the secreted protein IGFBP7.
      ,
      • Sasaki M.
      • Miyakoshi M.
      • Sato Y.
      • Nakanuma Y.
      Chemokine-chemokine receptor CCL2-CCR2 and CX3CL1-CX3CR1 axis may play a role in the aggravated inflammation in primary biliary cirrhosis.
      ,
      • Shimoda S.
      • Harada K.
      • Niiro H.
      • Yoshizumi T.
      • Soejima Y.
      • Taketomi A.
      • et al.
      Biliary epithelial cells and primary biliary cirrhosis: the role of liver-infiltrating mononuclear cells.
      ,
      • Tsuneyama K.
      • Harada K.
      • Yasoshima M.
      • Hiramatsu K.
      • Mackay C.R.
      • Mackay I.R.
      • et al.
      Monocyte chemotactic protein-1, -2, and -3 are distinctively expressed in portal tracts and granulomata in primary biliary cirrhosis: implications for pathogenesis.
      ,
      • Yasoshima M.
      • Kono N.
      • Sugawara H.
      • Katayanagi K.
      • Harada K.
      • Nakanuma Y.
      Increased expression of interleukin-6 and tumor necrosis factor-α in pathologic biliary epithelial cells: in situ and culture study.
      ]. Interestingly, when cultured cholangiocytes are treated with PAMPs, a treatment that reflects an activation of innate immunity, the cholangiocytes exhibit similar behaviors [
      • Harada K.
      • Ohba K.
      • Ozaki S.
      • Isse K.
      • Hirayama T.
      • Wada A.
      • et al.
      Peptide antibiotic human beta-defensin-1 and -2 contribute to antimicrobial defense of the intrahepatic biliary tree.
      ]. Thus, it is of interest to understand how “senescence” and “activated innate immunity” are related to each other.

      Innate immunity

      Activation of innate immunity

      Members of the TLR family are the best-characterized cell-surface receptors that recognize PAMPs, and, in humans, 10 TLRs (TLR1–10) have been identified [
      • Harada K.
      • Isse K.
      • Kamihira T.
      • Shimoda S.
      • Nakanuma Y.
      Th1 cytokine-induced downregulation of PPARγ in human biliary cells relates to cholangitis in primary biliary cirrhosis.
      ,
      • Harada K.
      • Ohira S.
      • Isse K.
      • Ozaki S.
      • Zen Y.
      • Sato Y.
      • et al.
      Lipopolysaccharide activates nuclear factor-kappaB through toll-like receptors and related molecules in cultured biliary epithelial cells.
      ,
      • Anderson K.V.A.
      Toll signaling pathways in the innate immune response.
      ,
      • Takeda K.
      • Akira S.
      Toll-like receptors in innate immunity.
      ]. In addition to immunocompetent cells, epithelial cells such as the intestinal epithelia express multiple TLRs. The activation of TLRs triggers an array of epithelial defense responses, including the production and release of antimicrobial peptides and cytokines/chemokines [
      • Harada K.
      • Nakanuma Y.
      Innate immunity in the pathogenesis of cholangiopathy: a recent update.
      ].

      Biliary innate immunity

      Similar to the intestinal mucosa, the biliary mucosa expresses multiple TLRs and intracellular adaptor molecules (myeloid differentiation factor 88 (MyD88) and receptor-associated kinase-1 (IRAK-1)), and the ligation of the receptors leads to intracellular signaling [
      • Harada K.
      • Nakanuma Y.
      Cholangiopathy with respect to biliary innate immunity.
      ,
      • Harada K.
      • Nakanuma Y.
      Innate immunity in the pathogenesis of cholangiopathy: a recent update.
      ]. For example, the response to LPS is mediated by an interaction with TLR4, which initiates the transduction of intracellular signals and this is followed by the activation of MyD88 and IRAK-1, resulting in the activation of NF-κB and then to the synthesis of antibiotics and also proinflammatory cytokines/chemokines [
      • Harada K.
      • Ohira S.
      • Isse K.
      • Ozaki S.
      • Zen Y.
      • Sato Y.
      • et al.
      Lipopolysaccharide activates nuclear factor-kappaB through toll-like receptors and related molecules in cultured biliary epithelial cells.
      ,
      • Anderson K.V.A.
      Toll signaling pathways in the innate immune response.
      ]. As part of a host’s defenses, cholangiocytes participate directly and actively in biliary innate immunity by secreting several antibiotics against bacteria [
      • Yasoshima M.
      • Kono N.
      • Sugawara H.
      • Katayanagi K.
      • Harada K.
      • Nakanuma Y.
      Increased expression of interleukin-6 and tumor necrosis factor-α in pathologic biliary epithelial cells: in situ and culture study.
      ,
      • Anderson K.V.A.
      Toll signaling pathways in the innate immune response.
      ] and also proinflammatory molecules such as IL-6, TNF-α, IL-8, CX3CL1, and CCL2 [
      • Tabibian J.H.
      • Trussoni C.E.
      • O’Hara S.P.
      • Splinter P.L.
      • Heimbach J.K.
      • LaRusso N.F.
      Characterization of cultured cholangiocytes isolated from livers of patients with primary sclerosing cholangitis.
      ,
      • Isse K.
      • Harada K.
      • Zen Y.
      • Kamihira T.
      • Shimoda S.
      • Harada M.
      • et al.
      Fractalkine and CX3CR1 are involved in the recruitment of intraepithelial lymphocytes of intrahepatic bile ducts.
      ,
      • Takeda K.
      • Akira S.
      Toll-like receptors in innate immunity.
      ,
      • Harada K.
      • Nakanuma Y.
      Innate immunity in the pathogenesis of cholangiopathy: a recent update.
      ,
      • Bals R.
      • Wang X.
      • Wu Z.
      • Freeman T.
      • Bafna V.
      • Zasloff M.
      • et al.
      Human β-defensin 2 is a salt- sensitive peptide antibiotic expressed in human lung.
      ,
      • Matsumoto K.
      • Fujii H.
      • Michalopoulos G.
      • Fung J.J.
      • Demetris A.J.
      Human biliary epithelial cells secrete and respond to cytokines and hepatocyte growth factors in vitro: interleukin-6, hepatocyte growth factor and epidermal growth factor promote DNA synthesis in vitro.
      ,
      • Chen X.M.
      • O’Hara S.P.
      • Nelson J.B.
      • Splinter P.L.
      • Small A.J.
      • Tietz P.S.
      • et al.
      Multiple TLRs are expressed in human cholangiocytes and mediate host epithelial defense responses to Cryptosporidium parvum via activation of NF-κB.
      ,
      • Harada K.
      • Isse K.
      • Nakanuma Y.
      Interferon γ accelerates NF-κB activation of biliary epithelial cells induced by Toll-like receptor and ligand interaction.
      ,
      • Shimoda S.
      • Harada K.
      • Niiro H.
      • Shirabe K.
      • Taketomi A.
      • Maehara Y.
      • et al.
      CX3CL1 (fractalkine): a signpost for biliary inflammation in primary biliary cirrhosis.
      ,
      • Okamura A.
      • Harada K.
      • Nio M.
      • Nakanuma Y.
      Participation of natural killer cells in the pathogenesis of bile duct lesions in biliary atresia.
      ,
      • Okamura A.
      • Harada K.
      • Nio M.
      • Nakanuma Y.
      Interleukin-32 production associated with biliary innate immunity and proinflammatory cytokines contributes to the pathogenesis of cholangitis in biliary atresia.
      ,
      • Harada K.
      • Nakanuma Y.
      Biliary innate immunity: function and modulation.
      ]. These chemokines can trigger the recruitment and activation of T cells, macrophages, neutrophils, and NK cells, followed by persistent periductal inflammation. In contrast to bacterial PAMPs, double-stranded RNA (dsRNA) is recognized by TLR3, and this is followed by the activation of interferon regulatory factor 3 (IRF-3) and NF-κB [
      • Harada K.
      • Sato Y.
      • Itatsu K.
      • Isse K.
      • Ikeda H.
      • Yasoshima M.
      • et al.
      Innate immune response to double-stranded RNA in biliary epithelial cells is associated with the pathogenesis of biliary atresia.
      ]. Collectively, the resultant periductal cytokine networks that are formed, contribute to biliary mucosal defense, and also the pathogenesis of fibrous cholangiopathy, and the subsequent acquired immunity [
      • O’Hara S.P.
      • Tabibian J.H.
      • Splinter P.L.
      • LaRusso N.F.
      The dynamic biliary epithelia: molecules, pathways, and disease.
      ,
      • Harada K.
      • Isse K.
      • Kamihira T.
      • Shimoda S.
      • Nakanuma Y.
      Th1 cytokine-induced downregulation of PPARγ in human biliary cells relates to cholangitis in primary biliary cirrhosis.
      ,
      • Harada K.
      • Ohba K.
      • Ozaki S.
      • Isse K.
      • Hirayama T.
      • Wada A.
      • et al.
      Peptide antibiotic human beta-defensin-1 and -2 contribute to antimicrobial defense of the intrahepatic biliary tree.
      ,
      • Harada K.
      • Ohira S.
      • Isse K.
      • Ozaki S.
      • Zen Y.
      • Sato Y.
      • et al.
      Lipopolysaccharide activates nuclear factor-kappaB through toll-like receptors and related molecules in cultured biliary epithelial cells.
      ,
      • Chen X.M.
      • O’Hara S.P.
      • Nelson J.B.
      • Splinter P.L.
      • Small A.J.
      • Tietz P.S.
      • et al.
      Multiple TLRs are expressed in human cholangiocytes and mediate host epithelial defense responses to Cryptosporidium parvum via activation of NF-κB.
      ,
      • Harada K.
      • Isse K.
      • Nakanuma Y.
      Interferon γ accelerates NF-κB activation of biliary epithelial cells induced by Toll-like receptor and ligand interaction.
      ]. A signaling pathway initiated by TLRs also involves activation of Ras, mediating the production of proinflammatory cytokines and chemokines by cholangiocytes such as IL6 followed by cholangiocyte proliferation [
      • O’Hara S.P.
      • Splinter P.L.
      • Trussoni C.E.
      • Gajdos G.B.
      • Lineswala P.N.
      • LaRusso N.F.
      Cholangiocyte N-Ras protein mediates lipopolysaccharide-induced interleukin 6 secretion and proliferation.
      ], and also inducing cellular senescence, as described below [
      • Tabibian J.H.
      • O’Hara S.P.
      • Splinter P.L.
      • Trussoni C.E.
      • LaRusso N.F.
      Cholangiocyte senescence by way of N-ras activation is a characteristic of primary sclerosing cholangitis.
      ]. These aspects of biliary innate immunity are shown in Fig. 2.
      Figure thumbnail gr2
      Fig. 2Innate immunity and physiopathological reactions in cholangiocytes. Activated innate immunity of cholangiocytes upon ligation of surface Toll-like receptors (TLRs) by pathogen-associated molecular patterns (PAMPs), induce several effector pathways. Representative effector: i) Persistent peribiliary fibroinflammation by secretion of chemokines and cytokines, ii) defense against bacterial and viral infection by IFN-β and antimicrobial peptides (defensin), iii) cellular senescence by activation of N-Ras (oncogene-induced senescence), iv) cell proliferation associated with IL-6, v) cell survive with features of “non-epithelial” cells and fibrosis, and vi) apoptosis induced by tumor factor-related apoptosis-inducing ligand (TRAIL) (EMT, epithelial-mesenchymal transition).

      Correlation between innate immunity and cellular senescence

      Recent studies using cultured cholangiocytes showed interesting findings connecting innate immunity to oncogene-induced cellular senescence [
      • Tabibian J.H.
      • O’Hara S.P.
      • Lindor K.D.
      Primary sclerosing cholangitis and the microbiota: current knowledge and perspectives on etiopathogenesis and emerging therapies.
      ,
      • Tabibian J.H.
      • Trussoni C.E.
      • O’Hara S.P.
      • Splinter P.L.
      • Heimbach J.K.
      • LaRusso N.F.
      Characterization of cultured cholangiocytes isolated from livers of patients with primary sclerosing cholangitis.
      ,
      • Tabibian J.H.
      • O’Hara S.P.
      • Splinter P.L.
      • Trussoni C.E.
      • LaRusso N.F.
      Cholangiocyte senescence by way of N-ras activation is a characteristic of primary sclerosing cholangitis.
      ,
      • O’Hara S.P.
      • Splinter P.L.
      • Trussoni C.E.
      • Gajdos G.B.
      • Lineswala P.N.
      • LaRusso N.F.
      Cholangiocyte N-Ras protein mediates lipopolysaccharide-induced interleukin 6 secretion and proliferation.
      ]. N-Ras is the predominant Ras isoform expressed in cholangiocytes. The short-term interaction of TLRs with LPS in cholangiocytes activate N-Ras and this results in the rapid phosphorylation of the downstream Ras effectors extracellular signal-regulated kinases (ERKs) 1 and 2 and the secretion of IL-6 [
      • O’Hara S.P.
      • Splinter P.L.
      • Trussoni C.E.
      • Gajdos G.B.
      • Lineswala P.N.
      • LaRusso N.F.
      Cholangiocyte N-Ras protein mediates lipopolysaccharide-induced interleukin 6 secretion and proliferation.
      ]. That is, a signaling pathway initiated by TLRs involving Ras mediates the production of proinflammatory cytokines and chemokines by cholangiocytes and the induction of cholangiocyte proliferation in response to microbial insult [
      • O’Hara S.P.
      • Splinter P.L.
      • Trussoni C.E.
      • Gajdos G.B.
      • Lineswala P.N.
      • LaRusso N.F.
      Cholangiocyte N-Ras protein mediates lipopolysaccharide-induced interleukin 6 secretion and proliferation.
      ].
      In contrast, persistent LPS treatment of cholangiocytes induced the expression of mediators of cellular senescence such as p16INK4a and p21WAF1/Cip in cholangiocytes [
      • Tabibian J.H.
      • O’Hara S.P.
      • Splinter P.L.
      • Trussoni C.E.
      • LaRusso N.F.
      Cholangiocyte senescence by way of N-ras activation is a characteristic of primary sclerosing cholangitis.
      ]. After an initial increase in Ki-67 mRNA expression, as mentioned above, this was followed by a progressive decrease in proliferation, consistent with a nonreplicative state. The percentage of SA-β-gal-positive cholangiocytes increased substantially among cholangiocytes treated with LPS, indicating an induction of cholangiocyte senescence. SASP components were also strongly induced in these cholangiocytes, indicating the progression of senescent cultured cholangiocytes to a SASP in vitro. Furthermore, N-Ras expression and activation peaked in LPS-treated cholangiocytes and remained elevated [
      • Tabibian J.H.
      • O’Hara S.P.
      • Splinter P.L.
      • Trussoni C.E.
      • LaRusso N.F.
      Cholangiocyte senescence by way of N-ras activation is a characteristic of primary sclerosing cholangitis.
      ], and this may have induced senescence (oncogene-induced cellular senescence) [
      • Tabibian J.H.
      • Trussoni C.E.
      • O’Hara S.P.
      • Splinter P.L.
      • Heimbach J.K.
      • LaRusso N.F.
      Characterization of cultured cholangiocytes isolated from livers of patients with primary sclerosing cholangitis.
      ,
      • Tabibian J.H.
      • O’Hara S.P.
      • Splinter P.L.
      • Trussoni C.E.
      • LaRusso N.F.
      Cholangiocyte senescence by way of N-ras activation is a characteristic of primary sclerosing cholangitis.
      ].
      Collectively, the aforementioned results revealed that N-Ras is activated in cholangiocytes following acute and also persistent LPS treatment. Whereas acute treatment induced the activation of innate immunity coupled with increased proliferative activities, persistent treatment triggered cellular senescence (Fig. 2). In chronic biliary diseases, the persistent process might predominate and the acute process could overlap with it.

      Cellular senescence and autophagy in fibrosing cholangiopathies

      While PBC, PSC, and BA belonging to fibrosing cholangiopathy [
      • 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.
      ,
      • EASL Clinical Practice Guidelines
      Management of cholestatic liver diseases.
      ] present distinct and unique pathological and immunological features, they also share common disease processes such as senescence, autophagy and inappropriate biliary innate immunity.

      Primary biliary cirrhosis

      A high prevalence of vaginal and urinary tract infections and the presence of bacterial components in bile and liver tissue and the molecular mimicry of human and bacterial pyruvate dehydrogenase complex-E2 (PDC-E2, a major epitope of AMAs), are reported in PBC [
      • Hirschfield G.M.
      • Gershwin M.E.
      The immunobiology and pathophysiology of primary biliary cirrhosis.
      ,
      • Hiramatsu K.
      • Harada K.
      • Tsuneyama K.
      • Sasaki M.
      • Fujita S.
      • Hashimoto T.
      • et al.
      Amplification and sequence analysis of partial bacterial 16S ribosomal RNA gene in gallbladder bile from patients with primary biliary cirrhosis.
      ,
      • Sasatomi K.
      • Noguchi K.
      • Sakisaka S.
      • Sata M.
      • Tanikawa K.
      Abnormal accumulation of endotoxin in biliary epithelial cells in primary biliary cirrhosis and primary sclerosing cholangitis.
      ,
      • Harada K.
      • Tsuneyama K.
      • Sudo Y.
      • Masuda S.
      • Nakanuma Y.
      Molecular identification of bacterial 16S ribosomal RNA gene in liver tissue of primary biliary cirrhosis: is Propionibacterium acnes involved in granuloma formation?.
      ,
      • Parikh-Patel A.
      • Gold E.B.
      • Worman H.
      • Krivy K.E.
      • Gershwin M.E.
      Risk factors for primary biliary cirrhosis in a cohort of patients from the United States.
      ,
      • Harada K.
      • Shimoda S.
      • Sato Y.
      • Isse K.
      • Ikeda H.
      • Nakanuma Y.
      Periductal interleukin-17 production in association with biliary innate immunity contributes to the pathogenesis of cholangiopathy in primary biliary cirrhosis.
      ]. Suggesting that the presence of microbes and the innate immune responses against them are also involved in the pathogenesis of PBC in addition to classical autoimmune features.

      Cellular senescence and SASP

      Cholangiocyte senescence exhibiting shortened telomeres, SA-β-gal expression, and augmented expression of p16INK4a and p21WAF1/Cip were reported frequently in CNSDC (Fig. 3), a which suggested that senescence might be involved in the formation of CNSDC and progressive bile duct loss in PBC [
      • Sasaki M.
      • Ikeda H.
      • Haga H.
      • Manabe T.
      • Nakanuma Y.
      Frequent cellular senescence in small bile ducts in primary biliary cirrhosis: a possible role in bile duct loss.
      ,
      • Sasaki M.
      • Ikeda H.
      • Yamaguchi J.
      • Nakada S.
      • Nakanuma Y.
      Telomere shortening in the damaged small bile ducts in primary biliary cirrhosis reflects ongoing cellular senescence.
      ]. While cellular senescence can be triggered by a number of stresses, oxidative stress is a potential causative factor in CNSDC in PBC [
      • Sasaki M.
      • Nakanuma Y.
      Biliary epithelial apoptosis, autophagy, and senescence in primary biliary cirrhosis.
      ,
      • Sasaki M.
      • Ikeda H.
      • Nakanuma Y.
      Activation of ATM signaling pathway is involved in oxidative stress-induced expression of mito-inhibitory p21WAF1/Cip1 in chronic non- suppurative destructive cholangitis in primary biliary cirrhosis: an immunohistochemical study.
      ,
      • Sasaki M.
      • Ikeda H.
      • Sato Y.
      • Nakanuma Y.
      Proinflammatory cytokine-induced cellular senescence of biliary epithelial cells is mediated via oxidative stress and activation of ATM pathway: a culture study.
      ,
      • Sasaki M.
      • Ikeda H.
      • Sato Y.
      • Nakanuma Y.
      Decreased expression of Bmi1 is closely associated with cellular senes-cence in small bile ducts in primary biliary cirrhosis.
      ]. For example, p21WAF1/Cip1 and an oxidative stress marker, 8-OHdG, were frequently and extensively coexpressed in the nuclei of CNSDC in PBC, and their expressions were correlated [
      • Sasaki M.
      • Ikeda H.
      • Nakanuma Y.
      Activation of ATM signaling pathway is involved in oxidative stress-induced expression of mito-inhibitory p21WAF1/Cip1 in chronic non- suppurative destructive cholangitis in primary biliary cirrhosis: an immunohistochemical study.
      ]. In addition, activation of biliary innate immunity by PAMPs is known to promote CX3CL1 and CX3CR1 expression in cholangiocytes followed by the recruitment of intraepithelial cytotoxic lymphocytes of CNSDC [
      • Acosta J.C.
      • O’Loghlen A.
      • Banito A.
      • Guijarro M.V.
      • Augert A.
      • Raguz S.
      • et al.
      Chemokine signaling via the CXCR2 receptor reinforces senescence.
      ], possibly via senescence as mentioned above [
      • Tabibian J.H.
      • O’Hara S.P.
      • Splinter P.L.
      • Trussoni C.E.
      • LaRusso N.F.
      Cholangiocyte senescence by way of N-ras activation is a characteristic of primary sclerosing cholangitis.
      ,
      • Yasoshima M.
      • Kono N.
      • Sugawara H.
      • Katayanagi K.
      • Harada K.
      • Nakanuma Y.
      Increased expression of interleukin-6 and tumor necrosis factor-α in pathologic biliary epithelial cells: in situ and culture study.
      ,
      • Harada K.
      • Ohira S.
      • Isse K.
      • Ozaki S.
      • Zen Y.
      • Sato Y.
      • et al.
      Lipopolysaccharide activates nuclear factor-kappaB through toll-like receptors and related molecules in cultured biliary epithelial cells.
      ,
      • O’Hara S.P.
      • Splinter P.L.
      • Trussoni C.E.
      • Gajdos G.B.
      • Lineswala P.N.
      • LaRusso N.F.
      Cholangiocyte N-Ras protein mediates lipopolysaccharide-induced interleukin 6 secretion and proliferation.
      ,
      • Harada K.
      • Shimoda S.
      • Sato Y.
      • Isse K.
      • Ikeda H.
      • Nakanuma Y.
      Periductal interleukin-17 production in association with biliary innate immunity contributes to the pathogenesis of cholangiopathy in primary biliary cirrhosis.
      ].
      Figure thumbnail gr3
      Fig. 3A hypothesis on the association of deregulated autophagy of cholangiocytes as autoimmune reaction of primary biliary cirrhosis. Autophagy is implicated in the intracellular antigen processing required for the presentation of major histocompatibility complex (MHC) class I and class II. Recycling of autolysosomes is deregulated, and this may be implicated in increased autophagosomes with increased expression of p62 and LC3 as well as disordered intracellular antigen processing with the presentation of MHC class I and class II. The cell-surface expression of mitochondrial antigen such as PDC-E2 and of MHC class I or class II molecules on cholangiocytes could lead to antigen presentation by MHC class I and II to immunocompetent cells such as dendritic cells, as well as to a direct target antigen recognition by PDC-E2-specific cytotoxic T cells, followed by autoimmune reactions.
      Most upregulated cytokines/chemokines in CNSDC are components of SASPs [
      • Sasaki M.
      • Ikeda H.
      • Haga H.
      • Manabe T.
      • Nakanuma Y.
      Frequent cellular senescence in small bile ducts in primary biliary cirrhosis: a possible role in bile duct loss.
      ,
      • Sasaki M.
      • Miyakoshi M.
      • Sato Y.
      • Nakanuma Y.
      Modulation of the microenvironment by senescent biliary epithelial cells may be involved in the pathogenesis of primary biliary cirrhosis.
      ,
      • Sasaki M.
      • Ikeda H.
      • Sato Y.
      • Nakanuma Y.
      Proinflammatory cytokine-induced cellular senescence of biliary epithelial cells is mediated via oxidative stress and activation of ATM pathway: a culture study.
      ,
      • Sasaki M.
      • Nakanuma Y.
      Novel approach to bile duct damage in primary biliary cirrhosis: participation of cellular senescence and autophagy.
      ], and senescent cholangiocytes are involved in modulating the periductal inflammatory microenvironment in CNSDC [
      • Sasaki M.
      • Ikeda H.
      • Sato Y.
      • Nakanuma Y.
      Proinflammatory cytokine-induced cellular senescence of biliary epithelial cells is mediated via oxidative stress and activation of ATM pathway: a culture study.
      ]. For example, the expression of CCL2 and CX3CL1 was substantially elevated in cholangiocytes in CNSDC in PBC, and their expression was colocalized with the expression of senescence markers. CCL2 and CX3CL1 produced by senescent cholangiocytes might promote infiltration by corresponding CCR2- and CX3CR1-expressing mononuclear cells and further aggravate cholangitis and eventual duct loss [
      • Sasaki M.
      • Miyakoshi M.
      • Sato Y.
      • Nakanuma Y.
      Chemokine-chemokine receptor CCL2-CCR2 and CX3CL1-CX3CR1 axis may play a role in the aggravated inflammation in primary biliary cirrhosis.
      ,
      • Sasaki M.
      • Kakuda Y.
      • Miyakoshi M.
      • Sato Y.
      • Nakanuma Y.
      Infiltration of inflammatory cells expressing mitochondrial proteins around bile ducts and in biliary epithelial layer may be involved in the pathogenesis in primary biliary cirrhosis.
      ]. These findings suggest that senescent cholangiocytes may participate in modulating, through their SASPs, the inflammatory microenvironment by recruiting monocytes and possibly other types of inflammatory cells.

      Progressive bile duct loss and senescence

      When cholangiocyte senescence occurs in injured bile ducts, the cholangiocytes are reported to remain in situ and not be replaced by normal cholangiocytes [
      • Sasaki M.
      • Nakanuma Y.
      Biliary epithelial apoptosis, autophagy, and senescence in primary biliary cirrhosis.
      ,
      • Sasaki M.
      • Ikeda H.
      • Haga H.
      • Manabe T.
      • Nakanuma Y.
      Frequent cellular senescence in small bile ducts in primary biliary cirrhosis: a possible role in bile duct loss.
      ,
      • Coppe J.P.
      • Patil C.K.
      • Rodier F.
      • Sun Y.
      • Munoz D.P.
      • Goldstein J.
      • et al.
      Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor.
      ,
      • Shelton D.N.
      • Chang E.
      • Whittier P.S.
      • Choi D.
      • Funk W.D.
      Microarray analysis of replicative senescence.
      ]. Therefore, the impaired replacement and nonproliferative properties of senescent cholangiocytes make them prone to further injuries, and this accentuates the inflammation caused by SASPs and is eventually followed by bile duct loss. Cellular senescence of the bile ductules that harbor hepatic stem/progenitor cells [
      • Sasaki M.
      • Ikeda H.
      • Haga H.
      • Manabe T.
      • Nakanuma Y.
      Frequent cellular senescence in small bile ducts in primary biliary cirrhosis: a possible role in bile duct loss.
      ] might also lead to impaired regeneration of cholangiocytes in PBC.

      Autophagy and bile duct pathology

      Recent studies have further demonstrated that autophagy is involved in the pathogenesis of hepatobiliary diseases [
      • Sasaki M.
      • Miyakoshi M.
      • Sato Y.
      • Nakanuma Y.
      Increased expression of mitochondrial proteins associated with autophagy in biliary epithelial lesions in primary biliary cirrhosis.
      ,
      • Sasaki M.
      • Miyakoshi M.
      • Sato Y.
      • Nakanuma Y.
      Autophagy mediates the process of cellular senescence char-acterizing bile duct damages in primary biliary cirrhosis.
      ,
      • Teckman J.H.
      • An J.K.
      • Blomenkamp K.
      • Schmidt B.
      • Perlmuter D.
      Mitochondrial autophagy and injury in the liver in alpha 1-antitrypsin deficiency.
      ].

      Autophagy and cholangiocytes

      Autophagy was specifically upregulated in CNSDC along with senescence in PBC [
      • Sasaki M.
      • Miyakoshi M.
      • Sato Y.
      • Nakanuma Y.
      Autophagy may precede cellular senescence of bile ductular cells in ductular reaction in primary biliary cirrhosis.
      ,
      • Sasaki M.
      • Miyakoshi M.
      • Sato Y.
      • Nakanuma Y.
      Autophagy mediates the process of cellular senescence char-acterizing bile duct damages in primary biliary cirrhosis.
      ] (Fig. 2). Vesicles positive for the autophagy marker, microtubule-associated protein-light chain 3β (LC3) [
      • Saitoh T.
      • Akira S.
      Regulation of innate immune responses by autophagy-related proteins.
      ], were observed to accumulate in the cytoplasm of damaged bile ducts expressing senescence markers in PBC [
      • Sasaki M.
      • Miyakoshi M.
      • Sato Y.
      • Nakanuma Y.
      Autophagy mediates the process of cellular senescence char-acterizing bile duct damages in primary biliary cirrhosis.
      ]; this suggested that autophagy could induce and facilitate cholangiocyte senescence [
      • White E.
      • Low S.W.
      Eating to exit: autophagy-enabled senescence revealed.
      ,
      • Young A.R.
      • Narita M.
      • Ferreira M.
      • Kirschner K.
      • Sadaie M.
      • Darot J.F.
      • et al.
      Autophagy mediates the mitotic senescence transition.
      ,
      • Sasaki M.
      • Ikeda H.
      • Nakanuma Y.
      Activation of ATM signaling pathway is involved in oxidative stress-induced expression of mito-inhibitory p21WAF1/Cip1 in chronic non- suppurative destructive cholangitis in primary biliary cirrhosis: an immunohistochemical study.
      ,
      • Collado M.
      • Blasco M.A.
      • Serrano M.
      Cellular senescence in cancer and aging.
      ,
      • Sasaki M.
      • Ikeda H.
      • Haga H.
      • Manabe T.
      • Nakanuma Y.
      Frequent cellular senescence in small bile ducts in primary biliary cirrhosis: a possible role in bile duct loss.
      ,
      • Sasaki M.
      • Miyakoshi M.
      • Sato Y.
      • Nakanuma Y.
      Modulation of the microenvironment by senescent biliary epithelial cells may be involved in the pathogenesis of primary biliary cirrhosis.
      ,
      • Yin X.M.
      • Ding W.X.
      • Gao W.
      Autophagy in the liver.
      ,
      • Sasaki M.
      • Nakanuma Y.
      Novel approach to bile duct damage in primary biliary cirrhosis: participation of cellular senescence and autophagy.
      ]. Furthermore, the aggregation of p62 was specifically increased coupled with the accumulation of LC3-positive vesicles in damaged bile ducts in PBC [
      • Sasaki M.
      • Ikeda H.
      • Yamaguchi J.
      • Nakada S.
      • Nakanuma Y.
      Telomere shortening in the damaged small bile ducts in primary biliary cirrhosis reflects ongoing cellular senescence.
      ]; p62 is an adaptor protein involved in the delivery of ubiquitin-bound cargo to autophagosomes [
      • Komatsu M.
      • Kurokawa H.
      • Waguri S.
      • Taguchi K.
      • Kobayashi A.
      • Ichimura Y.
      • et al.
      The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1.
      ]. The accumulation of p62 reflects deregulated autophagy with respect to insufficient processing of the damaged proteins that are bound to p62 [
      • Komatsu M.
      • Kurokawa H.
      • Waguri S.
      • Taguchi K.
      • Kobayashi A.
      • Ichimura Y.
      • et al.
      The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1.
      ]. Therefore, both the accumulation of LC3-positive vesicles and the aggregation of p62 could indicate deregulated autophagy in CNSDC in PBC, and deregulated autophagy might be involved in the induction of cholangiocyte senescence in several biliary diseases [
      • Sasaki M.
      • Miyakoshi M.
      • Sato Y.
      • Nakanuma Y.
      Autophagy may precede cellular senescence of bile ductular cells in ductular reaction in primary biliary cirrhosis.
      ,
      • Sasaki M.
      • Ikeda H.
      • Sato Y.
      • Nakanuma Y.
      Decreased expression of Bmi1 is closely associated with cellular senes-cence in small bile ducts in primary biliary cirrhosis.
      ,
      • Sasaki M.
      • Miyakoshi M.
      • Sato Y.
      • Nakanuma Y.
      Increased expression of mitochondrial proteins associated with autophagy in biliary epithelial lesions in primary biliary cirrhosis.
      ].

      Mitochondrial antigen expression and autophagy in cholangiocytes

      Because mitochondria represent a major target of autophagy, deregulated autophagy of mitochondria may be involved in the autoimmune pathogenesis that occurs in PBC. Previously, intense granular expression of PDC-E2, a mitochondrial protein and major autoantigen in PBC, was observed in CNSDC in PBC [
      • Sasaki M.
      • Miyakoshi M.
      • Sato Y.
      • Nakanuma Y.
      Increased expression of mitochondrial proteins associated with autophagy in biliary epithelial lesions in primary biliary cirrhosis.
      ]. Interestingly, the granular expression of PDC-E2 was closely related to or colocalized with the expression of the autophagy marker LC3. In a cell-culture study, the accumulation of LC3-expressing puncta colocalized with PDC-E2 was markedly increased in cultured cholangiocytes exposed to various stresses and, later, the cell-surface expression of PDC-E2 was also detected. This suggested that the autophagic vacuoles that accumulate as a result of deregulated autophagy are responsible for the granular expression and the subsequent cell-surface expression of PDC-E2 [
      • Sasaki M.
      • Miyakoshi M.
      • Sato Y.
      • Nakanuma Y.
      Increased expression of mitochondrial proteins associated with autophagy in biliary epithelial lesions in primary biliary cirrhosis.
      ], which might be closely related to autoimmune pathogenesis in PBC. Fig. 3 presents our hypothesis regarding the association of deregulated autophagy and autoimmune pathogenesis in bile duct lesions in PBC. Notably, autophagy has been implicated in the intracellular antigen processing required for the presentation of the major histocompatibility complex (MHC) class I and class II [
      • Nedjic J.
      • Aichinger M.
      • Emmerich J.
      • Mizushima N.
      • Klein L.
      Autophagy in thymic epithelium shapes the T-cell repertoire and is essential for tolerance.
      ,
      • Paludan C.
      • Schmid D.
      • Landthaler M.
      • Vockerodt M.
      • Kube D.
      • Tuschl T.
      • et al.
      Endogenous MHC class II processing of a viral nuclear antigen after autophagy.
      ,
      • Munz C.
      Antigen processing via autophagy–not only for MHC class II presentation anymore?.
      ]. Collectively, the cell-surface expression of PDC-E2 and of MHC class I or class II molecules on cholangiocytes could lead to antigen presentation by MHC class I and II to immunocompetent cells, as well as to a direct target antigen recognition by PDC-E2-specific cytotoxic T cells [
      • Nakanuma Y.
      • Zen Y.
      • Portman B.C.
      Diseases of the bile duct.
      ,
      • White E.
      • Low S.W.
      Eating to exit: autophagy-enabled senescence revealed.
      ,
      • Isse K.
      • Harada K.
      • Zen Y.
      • Kamihira T.
      • Shimoda S.
      • Harada M.
      • et al.
      Fractalkine and CX3CR1 are involved in the recruitment of intraepithelial lymphocytes of intrahepatic bile ducts.
      ,
      • Nakanuma Y.
      • Tsuneyama K.
      • Kono N.
      • Hoso M.
      • Van de Water J.
      • Gershwin M.E.
      Biliary epithelial expression of pyruvate dehydrogenase complex in primary biliary cirrhosis: an immunohistochemical and immunoelectron microscopic study.
      ].

      Primary sclerosing cholangitis

      The exact pathogenesis of the fibrosing cholangiopathy of PSC, which is frequently associated with inflammatory bowel disease [
      • Tabibian J.H.
      • O’Hara S.P.
      • Lindor K.D.
      Primary sclerosing cholangitis and the microbiota: current knowledge and perspectives on etiopathogenesis and emerging therapies.
      ,
      • Aron J.H.
      • Bowlus C.L.
      The immunobiology of primary sclerosing cholangitis.
      ], remains unclear. However, in PSC, an inappropriate innate immune response of the biliary tree directed against enteric microbes or their products, might be pathogenetically involved [
      • Tabibian J.H.
      • O’Hara S.P.
      • Lindor K.D.
      Primary sclerosing cholangitis and the microbiota: current knowledge and perspectives on etiopathogenesis and emerging therapies.
      ,
      • Aron J.H.
      • Bowlus C.L.
      The immunobiology of primary sclerosing cholangitis.
      ,
      • Nakanuma Y.
      • Harada K.
      • Katayanagi K.
      • Tsuneyama K.
      • Sasaki M.
      Definition and pathology of primary sclerosing cholangitis.
      ,
      • Szavay P.O.
      • Leonhardt J.
      • Czech-Schmidt G.
      • Petersen C.
      The role of reovirus type 3 infection in an established murine model for biliary atresia.
      ,
      • Matsushita H.
      • Miyake Y.
      • Takaki A.
      • Yasunaka T.
      • Koike K.
      • Ikeda F.
      • et al.
      TLR4, TLR9, and NLRP3 in biliary epithelial cells of primary sclerosing cholangitis: relationship with clinical characteristics.
      ,
      • O’Mahony C.A.
      • Vierling J.M.
      Etiopathogenesis of primary sclerosing cholangitis.
      ,
      • Eaton J.E.
      • Talwalkar J.A.
      • Lazaridis K.N.
      • Gores G.J.
      • Lindor K.D.
      Pathogenesis of primary sclerosing cholangitis and advances in diagnosis and management.
      ,
      • Broomé U.
      • Grunewald J.
      • Scheynius A.
      • Olerup O.
      • Hultcrantz R.
      • et al.
      Preferential V beta3 usage by hepatic T lymphocytes in patients with primary sclerosing cholangitis.
      ,
      • Schwarze C.
      • Terjung B.
      • Lilienweiss P.
      • Beuers U.
      • Herzog V.
      • Sauerbruch T.
      • et al.
      IgA class antineutrophil cytoplasmic antibodies in primary sclerosing cholangitis and autoimmune hepatitis.
      ,
      • Pokorny C.S.
      • Norton I.D.
      • McCaughan G.W.
      Selby WS Anti-neutrophil cytoplasmic antibody: a prognostic indicator in primary sclerosing cholangitis.
      ,
      • Terjung B.
      • Söhne J.
      • Lechtenberg B.
      • Gottwein J.
      • Muennich M.
      • Herzog V.
      • et al.
      P-ANCAs in autoimmune liver disorders recognise human beta-tubulin isotype 5 and cross-react with microbial protein FtsZ.
      ,
      • Eksteen B.
      • Grant A.J.
      • Miles A.
      • Curbishley S.M.
      • Lalor P.F.
      • Hübscher S.G.
      • et al.
      Hepatic endothelial CCL25 mediates the recruitment of CCR9+ gut-homing lymphocytes to the liver in primary sclerosing cholangitis.
      ]. However, the intestinal lymphocytes activated in gut-associated lymphoid tissues can also play key roles in PSC, in which hepatic expression of MAdCAM-1 and CCL25 may recruit these intestinal lymphocytes to the liver [
      • Eksteen B.
      • Grant A.J.
      • Miles A.
      • Curbishley S.M.
      • Lalor P.F.
      • Hübscher S.G.
      • et al.
      Hepatic endothelial CCL25 mediates the recruitment of CCR9+ gut-homing lymphocytes to the liver in primary sclerosing cholangitis.
      ,
      • Seidel D.
      • Eickmeier I.
      • Kühl A.A.
      • Hamann A.
      • Loddenkemper C.
      • Schott E.
      CD8 T cells primed in the gut-associated lymphoid tissue induce immune-mediated cholangitis in mice.
      ,
      • Trivedi P.J.
      • Adams D.H.
      Mucosal immunity in liver autoimmunity: a comprehensive review.
      ,
      • Bowlus C.L.
      Cutting edge issues in primary sclerosing cholangitis.
      ].

      Cellular senescence and SASP

      Recently, cholangiocytes of affected bile ducts in PSC were shown to exhibit senescence phenotypes [
      • Tabibian J.H.
      • Trussoni C.E.
      • O’Hara S.P.
      • Splinter P.L.
      • Heimbach J.K.
      • LaRusso N.F.
      Characterization of cultured cholangiocytes isolated from livers of patients with primary sclerosing cholangitis.
      ,
      • Tabibian J.H.
      • O’Hara S.P.
      • Splinter P.L.
      • Trussoni C.E.
      • LaRusso N.F.
      Cholangiocyte senescence by way of N-ras activation is a characteristic of primary sclerosing cholangitis.
      ]. Tabibian et al. demonstrated that the cholangiocytes of PSC livers overexpressed senescence-associated p16INK4a, and that cultured cholangiocytes derived from PSC explants expressed senescence markers such as SA-β-gal [
      • Tabibian J.H.
      • Trussoni C.E.
      • O’Hara S.P.
      • Splinter P.L.
      • Heimbach J.K.
      • LaRusso N.F.
      Characterization of cultured cholangiocytes isolated from livers of patients with primary sclerosing cholangitis.
      ]. The proliferation rate of the cultured cholangiocytes derived from PSC explants was low, which supported the occurrence of senescence. Moreover, SASP components were highly expressed in cholangiocytes cultured from PSC explants. These SASP components are presumed to be involved in the chemotaxis of inflammatory cells around bile ducts. For example, IL-6 can induce the proliferation of auto-reactive T cells and enhance immunoglobulin production, and T cells as well as substantial numbers of B cells are detected in the sclerosing cholangitis of PSC [
      • Nakanuma Y.
      • Zen Y.
      • Portman B.C.
      Diseases of the bile duct.
      ,
      • Aron J.H.
      • Bowlus C.L.
      The immunobiology of primary sclerosing cholangitis.
      ,
      • Nakanuma Y.
      • Harada K.
      • Katayanagi K.
      • Tsuneyama K.
      • Sasaki M.
      Definition and pathology of primary sclerosing cholangitis.
      ]. Other SASP components such as CCL2 and CX3CL1 might be involved in the periductal fibrosis of PSC [
      • Shimoda S.
      • Harada K.
      • Niiro H.
      • Shirabe K.
      • Taketomi A.
      • Maehara Y.
      • et al.
      Interaction between Toll-like receptors and natural killer cells in the destruction of bile ducts in primary biliary cirrhosis.
      ]. Furthermore, senescent cholangiocytes may acquire mesenchymal features followed by periductal fibrosis as reported in allograft rejection [
      • Brain J.G.
      • Robertson H.
      • Thompson E.
      • Humphreys E.H.
      • Gardner A.
      • Booth T.A.
      • et al.
      Biliary epithelial senescence and plasticity in acute cellular rejection.
      ,
      • Rygiel K.A.
      • Robertson H.
      • Marshall H.L.
      • Pekalski M.
      • Zhao L.
      • Booth T.A.
      • et al.
      Epithelial-mesenchymal transition contributes to portal tract fibrogenesis during human chronic liver disease.
      ]. Collectively, these findings raise the possibility that PSC cholangiocytes undergo sustained cellular senescence, and that SASPs, in turn, might contribute to fibrosing cholangiopathy in PSC.
      As described previously, cholangiocytes in which senescence is experimentally induced can, in turn, cause senescence in bystander cholangiocytes and other cells [
      • Sasaki M.
      • Miyakoshi M.
      • Sato Y.
      • Nakanuma Y.
      Modulation of the microenvironment by senescent biliary epithelial cells may be involved in the pathogenesis of primary biliary cirrhosis.
      ,
      • Sasaki M.
      • Nakanuma Y.
      Novel approach to bile duct damage in primary biliary cirrhosis: participation of cellular senescence and autophagy.
      ]. These processes can lead to the accumulation of senescent cholangiocytes in the biliary tree and amplify the SASP burden by means of paracrine signaling in PSC [
      • Tabibian J.H.
      • O’Hara S.P.
      • Splinter P.L.
      • Trussoni C.E.
      • LaRusso N.F.
      Cholangiocyte senescence by way of N-ras activation is a characteristic of primary sclerosing cholangitis.
      ]. Furthermore, these senescent cholangiocytes may relate to ductopenia in PSC as in PBC.

      Biliary innate immunity for induction of cellular senescence as a possible etiology of PSC

      Inappropriate innate immunity and the activation of oncogenes, particularly N-Ras, might be responsible for the induction of cholangiocyte senescence [
      • Marzioni M.
      • Glaser S.S.
      • Francis H.
      • Phinizy J.L.
      • LeSage G.
      • Alpini G.
      Functional heterogeneity of cholangiocytes.
      ,
      • Tabibian J.H.
      • Trussoni C.E.
      • O’Hara S.P.
      • Splinter P.L.
      • Heimbach J.K.
      • LaRusso N.F.
      Characterization of cultured cholangiocytes isolated from livers of patients with primary sclerosing cholangitis.
      ,
      • Tabibian J.H.
      • O’Hara S.P.
      • Splinter P.L.
      • Trussoni C.E.
      • LaRusso N.F.
      Cholangiocyte senescence by way of N-ras activation is a characteristic of primary sclerosing cholangitis.
      ,
      • O’Hara S.P.
      • Splinter P.L.
      • Trussoni C.E.
      • Gajdos G.B.
      • Lineswala P.N.
      • LaRusso N.F.
      Cholangiocyte N-Ras protein mediates lipopolysaccharide-induced interleukin 6 secretion and proliferation.
      ]. Activation of TLRs in cultured cholangiocytes from PSC explant livers by using PAMPs, particularly LPS, can induce senescence [
      • Tabibian J.H.
      • O’Hara S.P.
      • Splinter P.L.
      • Trussoni C.E.
      • LaRusso N.F.
      Cholangiocyte senescence by way of N-ras activation is a characteristic of primary sclerosing cholangitis.
      ,
      • O’Hara S.P.
      • Splinter P.L.
      • Trussoni C.E.
      • Gajdos G.B.
      • Lineswala P.N.
      • LaRusso N.F.
      Cholangiocyte N-Ras protein mediates lipopolysaccharide-induced interleukin 6 secretion and proliferation.
      ]. The PSC cholangiocytes showed a reversible increase in the expression of TLRs, particularly TLR4, and an activation of the MyD88/IRAK signaling complex, suggesting an association of excessive innate immune responses with PSC [
      • Eaton J.E.
      • Talwalkar J.A.
      • Lazaridis K.N.
      • Gores G.J.
      • Lindor K.D.
      Pathogenesis of primary sclerosing cholangitis and advances in diagnosis and management.
      ,
      • Broomé U.
      • Grunewald J.
      • Scheynius A.
      • Olerup O.
      • Hultcrantz R.
      • et al.
      Preferential V beta3 usage by hepatic T lymphocytes in patients with primary sclerosing cholangitis.
      ,
      • Schwarze C.
      • Terjung B.
      • Lilienweiss P.
      • Beuers U.
      • Herzog V.
      • Sauerbruch T.
      • et al.
      IgA class antineutrophil cytoplasmic antibodies in primary sclerosing cholangitis and autoimmune hepatitis.
      ,
      • Pokorny C.S.
      • Norton I.D.
      • McCaughan G.W.
      Selby WS Anti-neutrophil cytoplasmic antibody: a prognostic indicator in primary sclerosing cholangitis.
      ,
      • Terjung B.
      • Söhne J.
      • Lechtenberg B.
      • Gottwein J.
      • Muennich M.
      • Herzog V.
      • et al.
      P-ANCAs in autoimmune liver disorders recognise human beta-tubulin isotype 5 and cross-react with microbial protein FtsZ.
      ,
      • Eksteen B.
      • Grant A.J.
      • Miles A.
      • Curbishley S.M.
      • Lalor P.F.
      • Hübscher S.G.
      • et al.
      Hepatic endothelial CCL25 mediates the recruitment of CCR9+ gut-homing lymphocytes to the liver in primary sclerosing cholangitis.
      ,
      • Seidel D.
      • Eickmeier I.
      • Kühl A.A.
      • Hamann A.
      • Loddenkemper C.
      • Schott E.
      CD8 T cells primed in the gut-associated lymphoid tissue induce immune-mediated cholangitis in mice.
      ,
      • Trivedi P.J.
      • Adams D.H.
      Mucosal immunity in liver autoimmunity: a comprehensive review.
      ,
      • Bowlus C.L.
      Cutting edge issues in primary sclerosing cholangitis.
      ]. This endotoxin hyper-responsiveness was probably caused by the stimulatory effect exerted by IFN-γ and TNF-α, which are abundantly expressed in PSC livers and which enhance TLR4-mediated endotoxin signaling in cholangiocytes [
      • Mueller T.
      • Beutler C.
      • Picó A.H.
      • Shibolet O.
      • Pratt D.S.
      • Pascher A.
      • et al.
      Enhanced innate immune responsiveness and intolerance to intestinal endotoxins in human biliary epithelial cells contributes to chronic cholangitis.
      ]. TNF-α inhibition partly restored protective innate immune tolerance, which suggested that endogenous TNF-α secretion contributed to inappropriate endotoxin responses in cholangiocytes in PSC [
      • LaRusso N.F.
      • Shneider B.L.
      • Black D.
      • Gores G.J.
      • James S.P.
      • Doo E.
      • et al.
      Primary sclerosing cholangitis: summary of a workshop.
      ].
      As mentioned above, in cell-culture studies, Tabibian et al. found that cholangiocytes expressing active N-Ras showed an increased proportion of SA-β-gal-positive cells and increased secretion of IL-6, suggesting that LPS-induced activation of N-Ras induces the senescence and SASP [
      • O’Hara S.P.
      • Tabibian J.H.
      • Splinter P.L.
      • LaRusso N.F.
      The dynamic biliary epithelia: molecules, pathways, and disease.
      ,
      • Tabibian J.H.
      • O’Hara S.P.
      • Splinter P.L.
      • Trussoni C.E.
      • LaRusso N.F.
      Cholangiocyte senescence by way of N-ras activation is a characteristic of primary sclerosing cholangitis.
      ]. Interestingly, N-Ras and activated Ras were both expressed at substantially higher levels in cholangiocytes of PSC livers than of control livers [
      • O’Hara S.P.
      • Tabibian J.H.
      • Splinter P.L.
      • LaRusso N.F.
      The dynamic biliary epithelia: molecules, pathways, and disease.
      ,
      • Tabibian J.H.
      • O’Hara S.P.
      • Splinter P.L.
      • Trussoni C.E.
      • LaRusso N.F.
      Cholangiocyte senescence by way of N-ras activation is a characteristic of primary sclerosing cholangitis.
      ], which supports a potential role for activated N-Ras signaling in the induction of the cholangiocyte senescence and SASP of PSC.

      Autophagy and cellular senescence

      O’Hara et al. reported that lysosomal content was markedly increased in cultured senescent cholangiocytes after LPS treatment [
      • O’Hara S.P.
      • Splinter P.L.
      • Trussoni C.E.
      • Gajdos G.B.
      • Lineswala P.N.
      • LaRusso N.F.
      Cholangiocyte N-Ras protein mediates lipopolysaccharide-induced interleukin 6 secretion and proliferation.
      ]. Lysosomal increase is recognized to be associated with autophagy, and thus this phenomenon may indicate the participation of autophagy in cholangiocyte senescence [
      • Sasaki M.
      • Nakanuma Y.
      Biliary epithelial apoptosis, autophagy, and senescence in primary biliary cirrhosis.
      ]. Furthermore, PSC patients frequently present autoantibodies such as the perinuclear anti-neutrophil cytoplasmic antibody (pANCA) [
      • Schwarze C.
      • Terjung B.
      • Lilienweiss P.
      • Beuers U.
      • Herzog V.
      • Sauerbruch T.
      • et al.
      IgA class antineutrophil cytoplasmic antibodies in primary sclerosing cholangitis and autoimmune hepatitis.
      ,
      • Pokorny C.S.
      • Norton I.D.
      • McCaughan G.W.
      Selby WS Anti-neutrophil cytoplasmic antibody: a prognostic indicator in primary sclerosing cholangitis.
      ]. Interestingly, pANCAs appear to cross-react with β-tubular isotype 5 and with the bacterial cytoskeletal protein FxsZ, which is expressed by intestinal flora [
      • Terjung B.
      • Söhne J.
      • Lechtenberg B.
      • Gottwein J.
      • Muennich M.
      • Herzog V.
      • et al.
      P-ANCAs in autoimmune liver disorders recognise human beta-tubulin isotype 5 and cross-react with microbial protein FtsZ.
      ]. Thus, autophagy that is deregulated with respect to the recycling process used against microbial pathogens might be related to the occurrence of pANCAs in PSC, as speculated in PBC [
      • Sasaki M.
      • Nakanuma Y.
      Biliary epithelial apoptosis, autophagy, and senescence in primary biliary cirrhosis.
      ,
      • Sasaki M.
      • Miyakoshi M.
      • Sato Y.
      • Nakanuma Y.
      Autophagy may precede cellular senescence of bile ductular cells in ductular reaction in primary biliary cirrhosis.
      ,
      • Sasaki M.
      • Miyakoshi M.
      • Sato Y.
      • Nakanuma Y.
      A possible involvement of p62/sequestosome-1 in the process of biliary epithelial autophagy and senescence in primary biliary cirrhosis.
      ,
      • Sasaki M.
      • Miyakoshi M.
      • Sato Y.
      • Nakanuma Y.
      Increased expression of mitochondrial proteins associated with autophagy in biliary epithelial lesions in primary biliary cirrhosis.
      ,
      • Bansi D.S.
      • Fleming K.A.
      • Chapman R.W.
      Importance of antineutrophil cytoplasmic antibodies in primary sclerosing cholangitis and ulcerative colitis: prevalence, titre, and IgG subclass.
      ].

      Autoantibodies against cholangiocytes augment proinflammatory conditions of bile ducts

      PSC is considered an autoimmune-disease model based on the development of autoantibodies such as pANCAs and the selective destruction of bile ducts coupled with lymphoid cell infiltration. Karrar et al. [
      • Karrar A.
      • Broomé U.
      • Södergren T.
      • Jaksch M.
      • Bergquist A.
      • Björnstedt M.
      • et al.
      Biliary epithelial cell antibodies link adaptive and innate immune responses in primary sclerosing cholangitis.
      ] recently reported that biliary epithelial cell antibodies (BEC-Abs) were frequently detected in PSC patients, and that the stimulation of cholangiocytes with PSC IgGs containing BEC-Abs induced the expression of TLR4, TLR9, and MyD88 and the specific phosphorylation of ERK1/2 and the transcription factors ELK-1 and NF-κB. These autoantibodies also induce cholangiocytes to produce increased levels of IL-6 and the adhesion molecule CD44 [
      • Xu B.
      • Broome U.
      • Ericzon B.G.
      • Sumitran-Holgersson S.
      High frequency of autoantibodies in patients with primary sclerosing cholangitis that bind biliary epithelial cells and induce expression of CD44 and production of interleukin 6.
      ]. When TLR-expressing cholangiocytes are further stimulated with LPS and CpG DNA, they produce high levels of inflammatory cytokines/chemokines which in turn might lead to the recruitment of T cells, macrophages, and neutrophils. This raises the possibility that the adaptive immune response (BEC-Ab) elicited in PSC could facilitate the biliary innate immune response by inducing TLR expression in cholangiocytes. This chronic and amplified activation of cholangiocytes by BEC-Ab and also PAMPs might lead to persistent bile duct inflammation and the subsequent concentric fibrosing obliteration of the bile duct coupled with chemotactic periductal inflammatory cell infiltration [
      • Harada K.
      • Chiba M.
      • Okamura A.
      • Hsu M.
      • Sato Y.
      • Igarashi S.
      • et al.
      Monocyte chemoattractant protein-1 derived from biliary innate immunity contributes to hepatic fibrogenesis.
      ]. However, the target antigens of these BEC-Abs remain unknown.
      The pathogenesis of fibrosing cholangiopathy of PSC with respect to senescence and deregulated innate immunity is schematically shown in Fig. 4.
      Figure thumbnail gr4
      Fig. 4Possible pathogenesis of fibrosing cholangiopathy of primary sclerosing cholangitis with respect to cellular senescence and deregulated innate immunity. Cell injuries including enteric microbes or their products (PAMPs) and autoantibody against biliary epithelial cells (BEC-Ab) may be involved in the cellular senescence and senescence-associated secretory phenotype (SASP) and deregulated innate immunity. Senescent cholangiocytes and activated cholangiocytes by innate immunity may participate in modulation of the inflammatory microenvironment by secreting chemocytes, cytokines, growth factors, protease MMPs, and PAI-1 (SASP) followed by recruiting monocytes and possibly other types of inflammatory cells, induction of senescence in surrounding connective tissue cells (bystander effects) and progression of epithelial-mesenchymal transition (EMT) and fibrosis (PAMPs, pathogen-associated molecular patterns).

      Biliary atresia

      BA is characterized by a progressive sclerosing obliteration of extrahepatic bile ducts, though little is known about its pathogenesis. Although the autoimmune process might play a role in BA, particularly after virus clearance [
      • Mack C.L.
      • Falta M.T.
      • Sullivan A.K.
      • Karrer F.
      • Sokol R.J.
      • Freed B.M.
      • et al.
      Oligoclonal expansions of CD4+ and CD8+ T-cells in the target organ of patients with biliary atresia.
      ,
      • Selmi C.
      • Vergani D.
      • Mieli-Vergani G.
      Viruses and autoantibodies in biliary atresia.
      ,
      • Glaser S.S.
      Biliary atresia: is lack of innate immune response tolerance key to pathogenesis?.
      ], studies conducted using human materials and a virus-infected rodent model strongly suggest an association of Reoviridae possessing dsRNA and the deregulated innate immunity of cholangiocytes in the development of fibrosing cholangiopathy, particularly at the initial stage [
      • Szavay P.O.
      • Leonhardt J.
      • Czech-Schmidt G.
      • Petersen C.
      The role of reovirus type 3 infection in an established murine model for biliary atresia.
      ,
      • Harada K.
      • Sato Y.
      • Isse K.
      • Ikeda H.
      • Nakanuma Y.
      Induction of innate immune response and absence of subsequent tolerance to dsRNA in biliary epithelial cells relate to the pathogenesis of biliary atresia.
      ].

      Participation of innate immunity

      Tolerance to bacterial PAMPs is critical for maintaining homeostasis in the biliary tree [
      • Harada K.
      • Nakanuma Y.
      Innate immunity in the pathogenesis of cholangiopathy: a recent update.
      ,
      • Harada K.
      • Nakanuma Y.
      Biliary innate immunity: function and modulation.
      ]. Cholangiocytes possess an innate immune system consisting of TLRs, including TLR3, which recognize dsRNA. In addition, the cholangiocytes that line the remnants of extrahepatic bile ducts are reported to diffusely express TLR3 in BA [
      • Harada K.
      • Nakanuma Y.
      Cholangiopathy with respect to biliary innate immunity.
      ,
      • Harada K.
      • Sato Y.
      • Ikeda H.
      • Isse K.
      • Ozaki S.
      • Enomae M.
      • et al.
      Epithelial-mesenchymal transition induced by biliary innate immunity contributes to the sclerosing cholangiopathy of biliary atresia.
      ,
      • Mueller T.
      • Beutler C.
      • Picó A.H.
      • Shibolet O.
      • Pratt D.S.
      • Pascher A.
      • et al.
      Enhanced innate immune responsiveness and intolerance to intestinal endotoxins in human biliary epithelial cells contributes to chronic cholangitis.
      ,
      • Chuang J.H.
      • Chou M.H.
      • Wu C.L.
      • Du Y.Y.
      Implication of innate immunity in the pathogenesis of biliary atresia.
      ]. Interestingly, the innate immune tolerance of dsRNA tolerance is lacking in the cholangiocytes, and a sustained induction of the innate immune response results in chronic inflammation and bile duct destruction in BA [
      • Lazaridis K.N.
      • Strazzabosco M.
      • Larusso N.F.
      The cholangiopathies: disorders of biliary epithelia.
      ,
      • Harada K.
      • Nakanuma Y.
      Cholangiopathy with respect to biliary innate immunity.
      ,
      • Feldman A.G.
      • Mack C.L.
      Biliary atresia: cellular dynamics and immune dysregulation.
      ]. Thus, deregulated biliary innate immunity is highly involved in the progressive bile duct fibrosis and inflammation that occur at the beginning stage of the disease.

      Enhanced apoptosis, epithelial mesenchymal transition and periductal fibrosis

      Stimulation of cultured cholangiocytes with poly(I:C), an analog of Reoviridae, induced the activation of NF-κB and IRF-3, followed by the production of antiviral IFN-β1, and also enhanced apoptosis through the production of tumor factor-related apoptosis-inducing ligand (TRAIL) [
      • Harada K.
      • Nakanuma Y.
      Cholangiopathy with respect to biliary innate immunity.
      ,
      • Harada K.
      • Sato Y.
      • Ikeda H.
      • Isse K.
      • Ozaki S.
      • Enomae M.
      • et al.
      Epithelial-mesenchymal transition induced by biliary innate immunity contributes to the sclerosing cholangiopathy of biliary atresia.
      ]. In BA, the cholangiocytes lining the remnants of extrahepatic bile ducts showed an enhancement of the TRAIL and single-stranded DNA-positive apoptosis in conjunction with the activation of NF-κB and IRF-3, which suggested that the continuing apoptosis of cholangiocytes leads to biliary obliteration in BA [
      • Harada K.
      • Nakanuma Y.
      Cholangiopathy with respect to biliary innate immunity.
      ,
      • Harada K.
      • Sato Y.
      • Ikeda H.
      • Isse K.
      • Ozaki S.
      • Enomae M.
      • et al.
      Epithelial-mesenchymal transition induced by biliary innate immunity contributes to the sclerosing cholangiopathy of biliary atresia.
      ,
      • Harada K.
      • Ohira S.
      • Isse K.
      • Ozaki S.
      • Zen Y.
      • Sato Y.
      • et al.
      Lipopolysaccharide activates nuclear factor-kappaB through toll-like receptors and related molecules in cultured biliary epithelial cells.
      ]. Although the deregulated innate immune response to dsRNA reduced the viability of human cultured cholangiocytes in culture through TRAIL-mediated apoptosis, the rate of cell death was approximately 70% [
      • Harada K.
      • Nakanuma Y.
      Cholangiopathy with respect to biliary innate immunity.
      ,
      • Harada K.
      • Sato Y.
      • Ikeda H.
      • Isse K.
      • Ozaki S.
      • Enomae M.
      • et al.
      Epithelial-mesenchymal transition induced by biliary innate immunity contributes to the sclerosing cholangiopathy of biliary atresia.
      ].
      The cultured cholangiocytes that evaded apoptosis showed a gradual loss of the epithelial markers CK19 and E-cadherin, and increased expression of the mesenchymal marker S100A4 and a transcription factor essential for EMT, Snail; as a result of increased susceptibility to TGF-β1 and the production of bFGF, raised the possibility of the occurrence of EMT in cholangiocytes [
      • Harada K.
      • Nakanuma Y.
      Cholangiopathy with respect to biliary innate immunity.
      ,
      • Harada K.
      • Sato Y.
      • Ikeda H.
      • Isse K.
      • Ozaki S.
      • Enomae M.
      • et al.
      Epithelial-mesenchymal transition induced by biliary innate immunity contributes to the sclerosing cholangiopathy of biliary atresia.
      ,
      • Harada K.
      • Nakanuma Y.
      Innate immunity in the pathogenesis of cholangiopathy: a recent update.
      ]. In fact, mesenchymal markers and Snail were expressed but CK19 and E-cadherin were not expressed in the cholangiocytes lining the remnants of extrahepatic bile ducts in BA, suggesting that these cholangiocytes were undergoing EMT [
      • Harada K.
      • Sato Y.
      • Ikeda H.
      • Isse K.
      • Ozaki S.
      • Enomae M.
      • et al.
      Epithelial-mesenchymal transition induced by biliary innate immunity contributes to the sclerosing cholangiopathy of biliary atresia.
      ]. However, there have been several reports against EMT in the liver as a source of myofibroblasts, using lineage tracing studies [
      • Kisseleva T.
      • Brenner D.A.
      Is it the end of the line for the EMT?.
      ,
      • Chu A.S.
      • Diaz R.
      • Hui J.J.
      • Yanger K.
      • Zong Y.
      • Alpini G.
      • et al.
      Lineage tracing demonstrates no evidence of cholangiocyte epithelial-to-mesenchymal transition in murine models of hepatic fibrosis.
      ]. The surviving cholangiocytes expressing EMT markers on the remnants may produce the fibrogenic cytokine TGFβ [
      • Patsenker E.
      • Popov Y.
      • Stickel F.
      • Jonczyk A.
      • Goodman S.L.
      • Schuppan D.
      Inhibition of integrin alphavbeta6 on cholangiocytes blocks transforming growth factor-beta activation and retards biliary fibrosis progression.
      ], thus activating periductal fibroblasts followed by the accumulation of an extracellular matrix and progressive fibrosis. Collectively, these results suggest that these surviving cholangiocytes expressing EMT markers may directly or indirectly play a major role in the fibrosing cholangiopathy of BA [
      • Harada K.
      • Nakanuma Y.
      Cholangiopathy with respect to biliary innate immunity.
      ].

      Epithelial-mesenchymal transition and autophagy and senescence

      As discussed previously in acute allograft rejection, senescent cholangiocytes might be involved in the cholangiocytes expressing EMT markers resulting in fibrosing cholangiopathy in BA. Recently, cellular senescence markers were frequently detected in most of the Hering canals and in the interlobular bile ducts in end-stage BA [
      • Gutierrez-Reyes G.
      • del Carmen Garcia de Leon M.
      • Varela-Fascinetto G.
      • Valencia P.
      • Pérez Tamayo R.
      • Rosado C.G.
      • et al.
      Cellular senescence in livers from children with end stage liver disease.
      ], although the occurrence of BA in extrahepatic and large intrahepatic bile ducts, a main target of BA, has not been examined. Senescence or autophagy has been widely reported to be associated with developmental and also neoplastic EMT, and cancer cell invasion induced by the activation of EMT was recently reported to be associated with deregulated autophagy [
      • Gutierrez-Reyes G.
      • del Carmen Garcia de Leon M.
      • Varela-Fascinetto G.
      • Valencia P.
      • Pérez Tamayo R.
      • Rosado C.G.
      • et al.
      Cellular senescence in livers from children with end stage liver disease.
      ,
      • Karrar A.
      • Broomé U.
      • Södergren T.
      • Jaksch M.
      • Bergquist A.
      • Björnstedt M.
      • et al.
      Biliary epithelial cell antibodies link adaptive and innate immune responses in primary sclerosing cholangitis.
      ,
      • Chilosi M.
      • Carloni A.
      • Rossi A.
      • Poletti V.
      Premature lung aging and cellular senescence in the pathogenesis of idiopathic pulmonary fibrosis and COPD/emphysema.
      ]. An activation of TGF-β/Smad3-dependent signaling plays a key role in regulating autophagy-induced EMT [
      • Nitta T.
      • Sato Y.
      • Ren X.S.
      • Harada K.
      • Sasaki M.
      • Hirano S.
      • et al.
      Autophagy may promote carcinoma cell invasion and correlate with poor prognosis in cholangiocarcinoma.
      ].

      Fibrosing cholangiopathies share common disease process(es)

      PBC, PSC, BA and other fibrosing cholangiopathies present unique clin