Role of inflammation and proinflammatory cytokines in cholangiocyte pathophysiology

BBA - Molecular Basis of Disease 1864 (2018) 1270–1278

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Role of inflammation and proinflammatory cytokines in cholangiocyte pathophysiology☆

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Claudio Pinto, Debora Maria Giordano, Luca Maroni, Marco Marzioni⁎ Department of Gastroenterology and Hepatology, Università Politecnica delle Marche, Ancona, Italy

A R T I C L E I N F O

A B S T R A C T

Keywords: Cholangiocytes Inflammation PSC PBC Inflammasome

Cholangiocytes, the epithelial cells lining the bile ducts, are an important subset of liver cells. They are involved in the modification of bile volume and composition, and respond to endogenous and exogenous stimuli. Along the biliary tree, two different kinds of cholangiocytes exist: small and large cholangiocytes. Each type has different features and biological role in physiologic and pathologic conditions, and their immunobiology is important for understanding biliary diseases. Cholangiocytes provide the first line of defence against luminal microbes in the hepatobiliary system. Indeed, they express a variety of pattern recognition receptors and may start an antimicrobial defence activating a set of intracellular signalling cascades. In response to injury, cholangiocytes that are normally quiescent become reactive and acquire a neuroendocrine-like phenotype with the release of proinflammatory mediators and antimicrobial peptides, which support biliary epithelial integrity. These molecules act in an autocrine/paracrine manner to modulate cholangiocyte biology and determine the evolution of biliary damage. Failure or dysregulation of such mechanisms may influence the progression of cholangiopathies, a group of diseases that selectively target biliary cells. In this review, we focus on the response of cholangiocytes in inflammatory conditions, with a particular focus on the mechanism driving cholangiocytes adaptation to damage. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.

1. Introduction The biliary epithelium is composed of intrahepatic and extrahepatic bile ducts lined by cholangiocytes [1]. The human intrahepatic biliary epithelium is classified by size: hepatic ducts (> 800 μm), segmental ducts (400–800 μm), area ducts (300–400 μm), septal bile ducts (100 μm), interlobular ducts (15–100 μm), and bile ductules (< 15 μm) [2,3]. The intrahepatic biliary epithelium of rodents is formed by ducts of different sizes, small (< 15 μm in diameter) and large (> 15 μm) [4]. The cholangiocytes lining small and large bile ducts have been morphologically and functionally categorized into small and large cholangiocytes, respectively [5,6]. Small cholangiocytes are cuboidal and poorly specialized with a high nucleus/cytoplasm ratio, whereas large cholangiocytes are more columnar in shape and are supplied with more organelles and a small nucleus/cytoplasm ratio [7]. Under normal conditions, cholangiocytes actively participate to bile secretion via basal and hormone-regulated events [8,9]. Cholangiocytes represent only 3% to 5% of the total liver cell mass, but they are crucial for normal physiologic processes and contribute to multiple disease

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states of the biliary tract [1,10,11]. At level of the apical and basolateral membrane of cholangiocytes there are various transporters, such as the water channel aquaporin, the Na+-glucose transporter (SGLT1) and the Cl−/HCO3 exchanger (AE2). These molecules facilitate movement of water, electrolytes and solutes, modifying bile volume and composition. The functional impairment of these transporters could lead to cholestasis [12,13]. One of the most important and well-studied functions of cholangiocytes is secretin-induced release of bicarbonate into bile, which involves some of these transporters. The binding of secretin to the secretin receptor (SR) on the basolateral membrane of cholangiocytes leads to the formation of adenosine 3′,5′-cyclic monophosphate (cAMP) and to protein kinase A (PKA)-dependent phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) that is involved in the extrusion of Cl− in the lumen of bile ducts. The activation of the AE2, caused by the Cl− gradient across the plasma membrane, culminates in the net excretion of bicarbonate in bile [13], with passive efflux of water. As a result, cholangiocytes participate to up to 40% of the so-called bile salt—independent bile flow. Cholangiocytes also interact with resident and non-resident cells of

This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen. Corresponding author at: Department of Gastroenterology, Università Politecnica delle Marche, Nuovo Polo Didattico, III piano, Via Tronto 10, 60020 Ancona, Italy. E-mail address: [email protected] (M. Marzioni).

http://dx.doi.org/10.1016/j.bbadis.2017.07.024 Received 25 May 2017; Received in revised form 20 July 2017; Accepted 21 July 2017 Available online 25 July 2017 0925-4439/ © 2017 Elsevier B.V. All rights reserved.

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Fig. 1. Crosstalk between reactive cholangiocytes and liver cells during the course of cholangiopathies. Cholangiocytes respond to inflammatory insults by releasing a large amount of proinflammatory mediators, which act in autocrine and paracrine fashion and determine cholangiocytes proliferation, immune cells recruitment and myofibroblast differentiation.

duodenal microorganisms are believed to be a major source of microbial infection in several hepatobiliary diseases [21–23]. In addition, the portal vein constitutes an alternative source of infection for the biliary system [24,25]. To protect against the infection of pathogens, the biliary tract is endowed with several defence mechanisms. Cholangiocytes express a variety of pattern recognition receptors, such as Toll-like receptors (TLRs) [26–28]. Activation of pattern recognition receptorassociated intracellular signalling cascades results in the expression of different adhesion molecules, inflammatory mediators (such as cytokines/chemokines) and antimicrobial peptides, initiating epithelial immune responses against microbial infection [26–29]. Infection also induces signalling cascades required for maintenance of the biliary epithelial integrity and cross-talk with other immune cells in the liver, indicating a key role for cholangiocytes in the defence response in the biliary tree. Thus, the immunobiology of cholangiocytes is important for understanding diseases characterized by vanishing bile ducts, mainly primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC).

the bile ducts via pro-inflammatory and pro-fibrotic mediators, such as tumour necrosis factor α (TNF-α) and interleukin 6 (IL-6). Paracrine secretion of growth factors and peptides mediates extensive cross-talk with other liver cells, including hepatocytes, hepatic stellate cells (HSC), stem cells, subepithelial myofibroblasts, endothelial cells, and inflammatory cells [12–14]. Thus diseased cholangiocytes can sustain biliary inflammation and fibrosis. Moreover, cholangiocytes are involved in homeostasis maintenance of the biliary system via modulation of apoptosis (e.g. AkT1), senescence (e.g. N-RAS transforming protein), and proliferation (e.g. platelet-derived growth factor). In pathologic conditions, a damage to cholangiocytes may result in ductopenia, dysplasia, or malignant transformation of the bile ducts [12,13]. Another important concept that has emerged in the last years is the morphological and functional heterogeneity of cholangiocytes. Small and large cholangiocytes differentially express a wide array of receptors, enzymes and transporters. More is known about the function of large cholangiocytes, that are characterized by the expression of CFTR, AE2, bile acid transporters and SR, and proliferate in response to bile duct ligation. Functionally, large cAMP-dependent cholangiocytes are more susceptible to damage whereas small cholangiocytes are more resistant to liver injury [15,16]. Small cholangiocytes possess proliferative capabilities and display functional plasticity in disease, while large cholangiocytes are involved in hormone-regulated bile secretion. Moreover, there is evidence that stem cells that reside in the peribiliary glands can differentiate into cholangiocytes and may be involved in biliary remodelling and pathogenesis in the course of cholangiopathies [17,18]. Cholangiocytes differentially proliferate in response to liver injury/ toxins. Proliferation can be induced by multiple pathways and stimuli, including bile acids, acetylcholine, estrogen, hepatocyte growth factor, and IL-6. All these mediators act via the binding to specific receptors. Even some antiproliferative mediators are known such as somatostatin, gastrin and interferon γ [19,20]. Although human bile is sterile under normal conditions, the biliary tract opens into the duodenum, which is periodically colonized. Indeed,

2. Cholangiopathies The term cholangiopathies refers to a category of chronic liver diseases that share a central target: the cholangiocyte. Cholangiopathies can be sub-classified into immune-mediated, infectious, genetic, idiopathic, malignant, and secondary sclerosing cholangitis [12]. Each cholangiopathy has a unique manifestation and clinical course, yet they share cholangiocyte-mediated processes that contribute to their pathogenesis. Normally they involve proinflammatory signalling, innate immune responses, cholangiocyte proliferation and differentiation, as well as tissue repair processes. Thus, cholangiocytes are the targets but also contribute to disease development or recovery after injury. Various insults cause cholangiocyte activation in a process that leads to increased expression of proinflammatory cytokines and chemokines (e.g., IL-6, IL-8, TNF-α, and various growth factors) as well as activation of signalling cascades, such as Notch and Hedgehog [30]. The released 1271

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Fig. 2. Schematic illustration of the pathogenesis of cholangiopathies. In response to injury of different nature, cholangiocytes modify their phenotype, became reactive and secrete a variety of proinflammatory mediators. The balance between disease resolution, repair of damage, and perpetuation of the inflammatory state depends on genetic factors, epigenetic modifications and post-transcriptional phenomena. If the insult is not removed, chronic inflammation and cholangiocyte activation could result in fibrosis, ductopenia, cholestasis and malignant transformation of the biliary epithelium.

potentially, to devise novel effective therapies [35,36]. Because of their morbidity, mortality, need for liver transplantation, and overall cost to society, cholangiopathies are by now recognized as an important group of liver diseases. Most cholangiopathies display a progressive course leading to cirrhosis and liver failure, and the cause of most cholangiopathies remains obscure.

molecules act in autocrine and paracrine manners and ultimately lead to cholangiocyte proliferation, apoptosis, and senescence and may lead to local angiogenesis, fibrosis, and recruitment of innate and adaptive immune cells, mesenchymal cells, and endothelial cells (Fig. 1). Such events result in ductal reaction with an increase in the number of ductules, often accompanied by infiltration of leukocytes and lymphocytes, activation of liver progenitor cells, and an increase in matrix protein levels. Unless reversed, these events lead to periportal fibrosis, ductopenia and, eventually, biliary cirrhosis [31]. However, the same processes may also protect against further biliary insult or help repair injury to the biliary tree (Fig. 2). Generally, after an initial insult, cholangiocytes become activated and start to proliferate. This modification is functional to compensate for the anatomic loss of biliary cells and also to sustain their secretory activities [32]. However, biliary proliferation often decreases, and apoptotic mechanisms become prevalent with the development of ductopenia [13]. Along with proliferation, cholangiocyte response to injury is characterized by the so-called neuroendocrine-like transdifferentiation, which plays an essential role not only in sustaining biliary proliferation itself but also in immune responses, hepatic inflammation, and development of liver fibrosis [9,33]. In addition to local cholangiocyte-associated events, also genetic variants, epigenetic mechanisms and post-transcriptional phenomena (including the influence of microRNAs on protein expression [34]) may influence whether reactive cholangiocytes regress to a normal phenotype or lead to chronic inflammation of the bile duct, with progression of the cholangiopathy (Fig. 1). The influence of genetic factors in the pathophysiology of cholangiopathies is complex and the environmental contribution to disease progression is largely unknown, thus limiting our current understanding. Furthermore, each cholangiopathy likely has a heterogeneous pathogenesis, a variable natural history, and shows a different response to therapy. Thus, recognition of cholangiocytes heterogeneity along the biliary tree is necessary to better understand the cholangiopathies and,

3. Cytokines and cholangiocytes In the last 20–30 years, scientific advances have unveiled the crucial role of cholangiocytes in the immune pathogenesis of many hepatobiliary diseases. Biliary epithelial cells are quiescent in physiological conditions and become activated in response to stress or injury, starting to proliferate and secrete a number of peptides. The compensatory processes in response to hepatic insults include biliary hyperplasia, ductular reaction and lastly ductopenia, when apoptotic events prevail on proliferation. Hence, cholangiocytes are dynamic cells, which undergo extensive modification of their phenotype in response to liver inflammatory injury or exposure to bacterial and viral products. Activated biliary epithelial cells express a wide set of proinflammatory cytokines and chemokines, linked to ductular reaction (repair, inflammatory cell infiltration and myofibroblastic differentiation) (Fig. 1). The epithelial chemokines secreted can be transcytosed to the portal endothelium thereby recruiting circulating immune cells in the inflammation focus and attracting recruited cells to cholangiocyte surfaces. The type of chemokines and cytokines released depends on the triggering stimulus. In response to enteric-derived bacterial products, such as lipopolysaccharide (LPS), or IL-1 and TNF-α stimulation, cholangiocytes release in large amounts a variety of cytokines such as IL-8, the epithelial cell derived neutrophil activating protein (ENA-78) and growth-related oncogene (GRO), which determine neutrophil recruitment (Fig. 1) [21]. Conversely, biliary epithelial cells exposed to INFy show a switching in key inflammatory mediators released, from ones typical of acute inflammation to those characteristics of chronic 1272

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progression and irreversibility [56]. Collagen deposition is the result of chronic inflammation but in some pathological conditions such as congenital hepatic fibrosis (CHF), fibrosis seems to be caused by non-canonical inflammatory processes at the level of the portal space. A recent study carried out in mice lacking the polycystic kidney and hepatic disease 1 gene (PKHD1), as a model of CHF, demonstrated that fibrosis development is caused by cholangiocyte release of chemokines in a β-catenin-dependent manner, which determines macrophage chemotaxis and eventually leads to portal fibrosis. In particular, at early disease stage cholangiocytes release high amount of CXCL12 and CXCL5, while at later stages it has been observed the upregulation of CXCL1, CXCL10, CXCL2 and IL-1β. As a consequence, these molecules induce the recruitment of monocytes and macrophages, which start to release cytokines. In particular, M1 macrophages secrete TNF-α in the earlier phases of disease, that upregulates the expression of αvβ6 integrin on epithelial cells membrane leading to the binding and activation of latent TGF-β1. In this context, the proportion of myofibroblasts is low and it increases later during disease course, in parallel with the switching of macrophage state from M1 to M2. At this stage, the release of TGF-β1 becomes significant and results in collagen deposition into the peribiliary region [57]. Αvβ6 integrin has been shown to be overexpressed also at hepatic progenitor cells level during ductular reaction; it regulates not only the hepatic progenitor EpCAM+ formation and differentiation but also the development of fibrosis and hepatic tumorigenesis, even if the exact mechanism of αvβ6 integrin tumour promotion has to be further investigated [58]. Due to the strong association of hepatobiliary diseases with proinflammatory cytokine release, it is likely a role of these mediators on cholestasis development (Fig. 2). The hallmark of chronic cholangiopathies is cholestasis; however, the molecular mechanism of cholestasis development in these disorders is still poorly understood. In a study carried out in rat isolated bile duct unit (IBDU), a model of bile duct epithelium with conserved secretory ability, treatment with cytokines (TNF-α, IL-6, INFy and IL-1β) has been shown to reduce fluid secretion as well as to decrease the cAMP-dependent activity of CFTR and AE2 [59]. Moreover, normal rat cholangiocytes incubated with proinflammatory cytokines show a marked increase in paracellular transit [59]. Taken together these data suggest that proinflammatory mediators determine ductular cholestasis by impairing cAMP-dependent ion transport and the barrier functions of tight junction proteins, as shown by the increased permeability of biliary epithelia [59]. Proinflammatory cytokines determine also the release of nitric oxide (NO) by biliary epithelial cells via nitric oxide synthase-2 (NOS2) induction [59]. In cultured cholangiocytes, it has been demonstrated that NO production is stimulated by both TNF-α and INFy, and micromolar concentrations of NO impair cAMP-dependent fluid secretion and block Cl− and HCO3– transport by CFTR and AE2 exchanger, respectively [59]. Moreover, immunohistochemical data from stage III-IV PSC liver patients showed a marked expression of NOS2 in all biliary structures [59]. Collectively, these findings suggest a possible role of cytokinedependent NO-production and impairment of ion transport function in cholestasis development. As widely demonstrated, reactive cholangiocytes actively promote the ingravescence of liver injury by mediating the key features of biliary reaction such as duct injury and repair, immune cells infiltration, myofibroblastic differentiation of portal fibroblast and HSC (Fig. 2).

inflammatory process. In cholangiocytes, as well as in other human epithelial cells, it has been observed that INFy treatment reduces the release of IL-8 and enhances the secretion of the monocyte chemoattractant protein-1 (MCP-1) and other cytokines typical of a chronic inflammation, such as monokine induced by INFy (Mig), interferon inducible T cell alpha chemoattractant (ITAC) and interferon gamma inducible protein 10 (IP10) [21]. Furthermore, in response to IL-1β, TNF-α, IL-17 or TLRs activation, cholangiocytes release the macrophage inflammatory protein-3α (Mip-3a), a chemokine that induces dendritic cell chemotaxis [37]. Another chemokine released by reactive cholangiocytes is fractalkine or chemokine (C-XC-C motif) ligand 1 (CX3CL1), which belongs to CX3C family. This molecule acts as chemoattractant for monocytes and T lymphocytes in its soluble form, whereas it mediates immune cell-adhesion of leukocytes to cells expressing the CX3CR1 receptor in its cell-bound form (Fig. 1) [38]. Fractalkine seems to be involved in PBC pathogenesis, due to the presence of high levels of chemokine in sera of patients affected by PBC, which show a high infiltrate of CX3CR1-positive lymphocytes in portal tracts and in the biliary epithelium [39]. Cholangiocytes interact with a subset of CD4+ lymphocytes, the T helper (Th) 17 cells. The signal that leads to Th17 differentiation is given by IL-6 and IL-1β while IL-23 is necessary for subset maintenance [40,41]. Th17 cells infiltration has been observed in biopsy of patients with PBC in portal tract and in damaged interlobular bile ducts, but it is not yet clear how this subset of lymphocyte Th contributes to PBC [41]. Recruited leukocytes have a leading role in the protective immune response against biliary infection and participate in the pathological inflammatory reaction of bile ducts, like in PBC. In response to biliary injury, damaged cholangiocytes increase the levels of IL-6 and TNF-α compared to normal epithelial cells, which may act in autocrine or paracrine manner mediating different cellular events [42]. An in vitro study carried out on primary cultured cholangiocytes has shown that IL-6 treatment increases the proliferation of epithelial cells, highlighting its crucial role for biliary epithelial cells mass maintenance [43]. The effect of IL-6 on cell population subsistence has been observed in IL-6 deficient mice subjected to bile duct ligation, which showed a worst compensation of biliary cirrhosis compared to control [44]. IL-6 exerts also paracrine functions by promoting the terminal differentiation of B cells and the secretion of immunoglobulins [45]. TNF-α, on the other hand, plays a pleiotropic effect by increasing the expression of adhesion molecules on biliary epithelial cells membrane, in particular ICAM-1, MHC-I (overexpressed during CMV infection) and MHC-II, as well as by inducing the cytotoxic T lymphocytes functions [46]. Overexpression of MHC-II has been observed in damaged bile ducts of patient with allograft rejection, graft versus host disease, PBC and PSC [46–49]. Furthermore, TNF-α induces a direct bile duct damage in term of apoptosis as evidenced in cultured TNF-α treated rat hepatocytes [50]. During persistent liver injury, cholangiocytes synthetize and release TGF-β; in particular, β2 transcripts were detected at high levels in reactive bile ducts of fibrotic livers [51]. TGF-β reduces the expression of adhesion molecules on cell surfaces, promotes myofibroblastic differentiation of portal fibroblasts and hepatic stellate cells (HSC) [52] and inhibits proliferation in primary human cholangiocytes [53]. Moreover, this mediator induces cholangiocytes secretion of endothelin-1 and regulates, in a paracrine manner, the deposition of extracellular matrix in the adjacent mesenchymal cells [51]. In this context, biliary epithelial cells upregulate MCP-1 secretion, and the degree of upregulation is proportional to disease severity [54]. In particular, MCP-1 induces myofibroblastic differentiation and releasing of collagen-1 by portal fibroblast [55]. In the setting of ductular reaction, recent studies have revealed the importance of a member of the lysyl oxidase family (LOX), named LOX2, in the advanced phases of fibrosis. In particular, LOX2 is involved in extracellular matrix stabilization through collagen crosslinking, which stimulates HSC and portal fibroblast activation, as well as fibrosis

4. PAMPs, microbiota and cholangiocytes In recent years, increasing evidence supports the role of the enteric microbiota as a potentially key mediator of liver disease initiation and progression, such as in PSC and PBC. Even if the gut barrier limits bacterial translocation of microbial products, small quantities of these compounds reach the liver via the portal circulation. In physiological conditions, the mechanism of immune tolerance inhibits the excessive 1273

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benefit of oral antibiotics in the treatment PSC patients [74–76]. Recently our research group carried out in vitro and in vivo experiments to evaluate the role of Nlrp3 inflammasome activation in the biological response of cholangiocytes to injury [77]. We demonstrated an increased expression of the Nlrp3 inflammasome in both mice subjected to DDC diet, as an in vivo model of PSC, and in liver section from PSC patients as compared to relative controls. In vitro the activation of Nlrp3 leads to the upregulation of the proinflammatory cytokine IL-18. This increase is abolished in cultured cholangiocytes knocked-down for Nlrp3 gene expression, confirming the direct role of Nlrp3 inflammasome activation in IL-18 secretion. Our in vitro and in vivo experimental findings point out that Nlrp3 activation had no or minimal effect on cholangiocytes proliferation whereas had important effect on celladhesion molecules involved in epithelial barrier function. In particular, we have shown that in vitro activation of Nlrp3 inflammasome induces the downregulation of protein expression of both cell-adhesion markers, E-cadherin and Zonulin-1, and increases epithelial permeability on a functional level. The protein E-cadherin has been also evaluated in murine model of PSC, where its increased expression has been observed in bile ducts of Nlrp3−/− mice compared to wild-type controls. These findings were supported by an upregulation of E-cadherin gene expression in DDC-treated Nlrp3−/− mice compared to control group. Taken together these data suggest that the activation of Nlrp3 inflammasome mediated by PAMPs alters the epithelial barrier function of biliary cells, supporting the hypothesis that microbial products present in the bile could affect the biology of reactive biliary cells [77]. A number of other studies support the crucial role of the microbiota in liver disease onset. As an example, PBC patients show autoantibodies that cross-react with bacterial antigens from E. coli and N. aromaticivorans. In addition, PBC-cholangiocytes show abnormal accumulation of LPS in vivo [23]. Based on the data collected up to now, the alteration of physiological gut-liver axis may explain the development, progression and outcome of different hepatobiliary diseases.

activation of immune system against exogenous insults. However, under pathological conditions such as impairment of gut barrier function and modification of gut microbiota composition and potential dysbiosis, the translocation of bacteria or fragments of bacteria from intestine to liver can trigger hepatic inflammation and fibrosis [60]. Especially hepatocytes and Kuppfer cells are responsible for the metabolism and clearance of bacterial products in the liver but also cholangiocytes are able to recognize gut bacterial products. Indeed, pathogen-associated molecular patterns (PAMPs) present in the bile can be sensed through the related pattern recognition receptors. The best characterized of these families is the Toll-like receptors (TLRs) family and, to a lesser extent, the nucleotide binding oligomerization domain (NOD)-like receptors (NLRs) family [61]. In mammalian cells, 13 TLR receptors have been identified, whereas only 10 are expressed in humans. The TLR family is a set of membrane-spanning receptors known as mediators of innate immune system. Following PAMPs recognition, TLRs adaptor proteins are recruited to receptor complex, forming the so-called “signalosome” [62,63], thereby triggering several downstream events such as biosynthesis of proinflammatory cytokines/chemokines as well as recruitment of immune cell responses. The two major signalling cascades activated through TLRs members' receptors are the nuclear factor kappa-B (Nf-kB) and the mitogen-activated protein kinase (MAPK) pathways [64]. The interaction between LPS and TLR4 leads to cholangiocyte release of a broad spectrum of proinflammatory cytokines, such as IL-1β, IL-8, IL-6, MCP-1, TNF-α, INFy and TGF-β [65]. These stimuli enhance liver injury, immune cells infiltration, and cause a worsening of liver injury with induction of hepatic fibrosis. A study carried out on CFTR-KO mice subjected to dextran sulfate‑sodium (DSS) colitis model has shown the correlation between the defective channel function of CFTR and an increased TLR4mediated inflammatory response. Thus, cholangiocytes isolated from CFTR-KO mice and exposed to LPS showed an increase in TLR4-dependent cytokines secretion via Nf-kB pathway compared to controls [66]. Recent data indicate that CFTR is localized on cholangiocytes apical membrane in association with a multiprotein complex which acts as a negative regulator of non-receptor tyrosine kinases belonging to the Rous sarcoma oncogene cellular homolog (Src) family (SFKs). These family members are involved in different functions such as the phosphorylation of the cytoplasmic domain of TLR4, which leads to activation of the downstream signalling cascade. It has been shown that the reduced expression of CFTR determines a lower inhibition of Src tyrosin kinase, which self-activates and phosphorylates TLR4 cytoplasmic domain leading to an increase inflammatory response to endotoxin [67]. The NLR family includes a group of cytoplasmic soluble proteins (22 identified in humans) that detect intracellular pathogens or endogenous dangerous signals through damage-associated molecular patterns (DAMPs) [61]. A subgroup of NLRs is able to assemble into the inflammasome, a cytosolic multiprotein complex including a sensor protein, adaptor protein and an effector enzyme that can trigger hepatic inflammation. Recently, the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, pyrin domain-containing protein 3 (Nlrp3) has been described as member of innate immune system able to recognize both PAMPs and DAMPs [68]. Nlrp3 inflammasome activation determines caspase-1 activation and the secretion of mature proinflammatory IL-1β and IL-18 via NF-kB pathway [69]. A strong correlation between biliary epithelial cells and enteric-derived microbial components has been underlined in PSC, which is associated with inflammatory bowel disease, a disorder characterized by dysbiosis [70]. In line with the hypothesis of gut microbiota as a possible trigger of hepatobiliary disease, portal bacteremia, bactero-bilia [71,72] and 16s ribosomal Ribonucleic acid (rRNA) in bile are features described in course of PSC [22]. In addition, PSC-cholangiocytes accumulate in vivo high quantity of LPS, and in vitro show an increase response and loss of tolerance against the endotoxin [23,73]. Further evidence for a hypothetical etiological role of gut microbiota in the pathogenesis of PSC is provided by the possible therapeutic

5. Inflammation and cholangiocarcinogenesis Despite epidemiological data show a global increase in incidence and mortality of cholangiocarcinoma (CCA) in the last years, the molecular mechanisms involved in the development of this malignancy remain to be fully defined yet. CCA is an aggressive tumour composed of cells exhibiting a phenotype of biliary epithelial cells, but also nontumour cells like infiltrating lymphocytes (TIL), tumour associated macrophages (TAM), and cancer associated fibroblast (CAF) that play an important role in promoting malignant cell dissemination [78]. The major risk factor for CCA development is chronic biliary inflammation. The correlation between inflammation and cancer has been known since 1863, when Rudolf Virchow observed the presence of an inflammatory cells infiltrate in tumour tissues [79]. It is now well known that proinflammatory cytokines and other molecules released during chronic inflammation promote neoplastic transformation through damage of protoncogenes and tumour suppressor genes involved in cell growth, apoptosis, invasiveness and angiogenesis. As a result, cells undergo uncontrolled proliferation and acquire an invasive phenotype (Fig. 3). Landskron et al. recently summarized the major cytokines and the relative pathways involved in this processes [80]. The first step of malignant transformation is the acquisition of autonomous proliferation capability associated to apoptosis resistance. Due to overexpression of the mitogenic cytokine IL-6 during chronic inflammation of biliary tree, it is not surprisingly its possible role in cholangiocarcinogenesis. This concept is supported by the increasing levels of IL-6 detected in serum and bile of patients with CCA and in cultures of CCA cell lines [81]. IL-6 promotes survival of transformed cholangiocytes through different pathways. Among these, the p44/42 and p38 MAPKs have been largely characterized. In particular, the activation of p38 pathway by IL-6 determines a downregulation of a key gene regulator of cell cycle, 1274

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Fig. 3. Role of the cytokines in cholangiocarcinoma development. (a) In response to tissue insult or infection, cholangiocytes become active and start to release proinflammatory mediators, such as IL-6, TNF-α and IL-8. These cytokines induce cholangiocytes proliferation and immune cells recruitment to the injury site. (b) If injury persists, chronic inflammation is established. In this setting, proinflammatory cytokines like TNF-α can induce DNA damage by RONS generation as well as IL-6. This soluble mediator can induce tumorigenesis through epigenetic modification at the promoter level of oncogenes. Moreover, it plays a role in tumour growth by activating the anti-apoptotic gene Mcl-1. TGF-β can promote malignant EMT characterized by loss of structures involved in cell-cell adhesion. (c) Tumour invasion and neovascularization are induced by proinflammatory cytokines that support cell proliferation, apoptotic resistance, EMT, invasion (secretion of metalloproteinasis-2 or MMP2 by macrophages) and angiogenesis (VEGF and IL-8). (d) The metastatic process is favoured by tumour microenvironment, formed by immune cells infiltrating tumour (TAM, TIL and CAF).

p21WAF/CIP1, and promotes anchorage-independent cells growth conversely to the activation of p44/42 MAPK pathway that leads to anchorage-dependent cells growth [82]. Another mechanism by which IL-6 sustains tumour growth is the DNA methylation in the promoter region of target genes involved in cancer growth, such as the epidermal growth factor receptor (EGFR). In particular, in an in vitro experiment conducted on CCA cells line, it has been observed that overexpression of IL-6 reduces methylation of the EGFR promoter and increases both gene and protein expression [83]. In physiological condition, the binding between IL-6 and its receptor culminates with the translocation of activated STAT-3 to the nucleus and in the expression of IL-6 target genes. This event is under negative control of suppressor of cytokine signalling-3 (SOCS3). Recently, it has been described a second wave of activation of STAT-3 that evades from negative check of SOCS3 mediated by activated-EGFR [84]. IL-6 plays a role also in the apoptotic process of transformed biliary cells. A number of studies have demonstrated the upregulation of the antiapoptotic myeloid cell leukemia-1 (Mcl-1) mediated by IL-6. This process is induced by increasing expression of STAT-3 that leads to Mcl-1 transcription, enhancing cells survival [85]. Also TNF-α belongs to cytokines that exert growth factor activity. Silencing of TNF-α gene expression reduces both cell proliferation and invasion, via TNF-α/NF-kB/Akt/Bcl-2 pathway, in an autocrine fashion. These data are in line with previous reports showing that NFkB signalling promotes malignant cell proliferation in response to inflammatory stimuli, such as LPS [86]. A proposed mechanism through which TNF-α leads to promotion of a cancer phenotype is based on DNA damage induced by generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) that interfere with DNA repair [87,88]. In addition TNF-α can induce cell proliferation through indirect

mechanisms. Indeed, TNF-α acts on CD4+ lymphocytes to secrete IL-17 that seems to be involved in different types of cancer. In particular, an in vivo study carried out in mice lacking IL-17 showed a diminished incidence and size of tumour [89]. The role of IL-17 as well as other cytokines like IL-23 in tumour growth has been observed in a murine model of melanoma by activation of IL-6/STAT3 pathway [90]. Another important cytokine that regulates cell processes as growth, differentiation, immunity, apoptosis and survival is TGF-β. Following malignant transformation, TGF-β shifts its function from a tumour suppressor molecule, in early phases of tumorigenesis, to one promoting proliferation, survival and metastasis in the advances stages [91,92]. It has been shown that mutation of TGF-β receptor with the alteration of intracellular signalling mediators (such as SMAD4) and upregulation of cyclin D1 determine the resistance of CCA cells to the inhibitory effects of TGF-β [93]. SMAD4 is a tumour suppressor gene belonging to TGF-β pathway; its loss of expression has been described in extrahepatic biliary malignancy [94]. Another tumour suppressor gene that acts synergically with SMAD4 is PTEN. Mouse models lacking the expression of these two genes develop cholangiocarcinoma [95]. TGF-β has been also implicated in the epithelial mesenchymal transition (EMT), a process that leads to loss of structures involved in cell-cell adhesion and cytoskeletal reorganization and consequently to the increase of cell motility and invasion. The EMT process is driven by a set of embryonic transcription factors, including Snail (Snail1), Slug (Snail2), Twist1/2 and ZEB1/2, which repress the expression of cytokeratins and various junction proteins, principally E-cadherin. High TGF-β transcript variant 1 levels correlate with tumour recurrence, lymph node invasion and distant metastasis in iCCA patients [96]. According to this finding, the 1275

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6. Conclusions Cholangiocyte adaptation to injury in the setting of acute and chronic inflammation is a deeply complex and tightly regulated process. Intense research of the last decade has helped in shedding some light in the mechanisms involved in cholangiocyte response to injury. As we deepen our understanding of biliary pathophysiology, it is increasingly clear that cholangiocytes are not only the passive target of biliary diseases but actively contribute to disease resolution or progression. The understanding of biliary pathophysiology is likely to open new venues in the treatment of cholangiopathies by the specific targeting of intracellular pathways that modulate proliferation, cytokine production and interactions with other liver cells. Transparency Document The Transparency document associated with this article can be found, in online version. 1276

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