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How the biliary tree maintains immune tolerance?
Accepted Manuscript How the biliary tree maintains immune tolerance?
Haiyan Zhang, Patrick S.C. Leung, M. Eric Gershwin, Xiong Ma PII: DOI: Reference:
S0925-4439(17)30301-0 doi: 10.1016/j.bbadis.2017.08.019 BBADIS 64869
To appear in: Received date: Revised date: Accepted date:
15 May 2017 3 August 2017 9 August 2017
Please cite this article as: Haiyan Zhang, Patrick S.C. Leung, M. Eric Gershwin, Xiong Ma , How the biliary tree maintains immune tolerance?, (2017), doi: 10.1016/ j.bbadis.2017.08.019
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How the biliary tree maintains immune tolerance?
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Haiyan Zhang1, Patrick S.C.Leung2, M. Eric Gershwin2 and Xiong Ma1
Division
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Gastroenterology and
Hepatology,
Key Laboratory of
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Affiliations
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Gastroenterology and Hepatology, Ministry of Health, State Key Laboratory for Oncogenes and Related Genes, Renji Hospital, School of Medicine, Shanghai
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Jiao Tong University; Shanghai Institute of Digestive Disease; 145 Middle Shandong Road, Shanghai 200001, China Division of Rheumatology, Allergy, and Clinical Immunology, University of
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California at Davis, Davis, California, USA
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Correspondence:
Xiong Ma, MD, Ph.D. Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Institute of Digestive Disease, 145 Middle Shandong road, Shanghai 200001, China. Email: [email protected];
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ACCEPTED MANUSCRIPT Keywords: Cholangiocytes; biliary tree; immune tolerance; primary biliary cholangitis; primary sclerosing cholangitis; biliary atresia
Conflict of interest The authors declare no financial conflict of interest exists
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Financial Support
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This work was supported by awards from the National Natural Science
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NU
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Foundation of China (#81325002 and 81620108002 to Xiong Ma)
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ACCEPTED MANUSCRIPT Abbreviations:
Dendritic Cells
NKT cells
Natural Killer T cells
BECs
Biliary Epithelial Cells
LPS
Lipopolysaccharide
PAMPs
Pathogen Associated Molecular Patterns
PRRs
Pattern-Recognition Receptors
TLRs
Toll-Like Receptors
SV40
Simian Virus 40
MyD88
Myeloid Differentiation Factor 88
IRAK-1
IL-1 Receptor-Associated Kinase-1
IFN-γ
Interferon- γ
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DCs
RIG-1
Retinoic acid Induced Protein 1 Melanoma Differentiation Gene-5 Human β-defensins
hBD
Monocyte Chemotatctic Protein-1
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MCP-1
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CX3CL1 CX3CR1 SIgA
PSC AILD PD
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PBC
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CMV GVHD
Associated
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MDA-5
Fractalkine CX3CL1 Receptor Secretory IgA Cytomegalovirus Graft Versus Host Disease Primary Biliary Cholangitis Primary Sclerosing Cholangitis Autoimmune Liver Diseases Programmed-death
TRAIL
TNF-related Apoptosis-inducing Ligand
PPARγ RRV
Peroxisome Receptor γ Rhesus Rotavirus
EMT
Epithelial-Mesenchymal Transition
ANA
Antinuclear antibodies
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Proliferator-activated
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Perinuclear anti-neutrophil cytoplasmic
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antibody
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ACCEPTED MANUSCRIPT Research Highlights 1. BECs are the first line of defense of the biliary tree against foreign substances. 2. BECs play important roles in maintaining tolerance through various immunological pathways.
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3. Breach of tolerance in the biliary tree results in various cholangiopathies.
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Abstract The liver is a vital organ with distinctive anatomy, histology and heterogeneous cell populations. These characteristics are of particular importance in maintaining immune homeostasis within the liver microenvironments, notably the biliary tree. Cholangiocytes are the first line of defense of the biliary tree against foreign
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substances, and are equipped to participate through various immunological pathways. Indeed, cholangiocytes protect against pathogens by TLRs-related
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signaling; maintain tolerance by expression of IRAK-M and PPARγ; limit immune
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response by inducing apoptosis of leukocytes; present antigen by expressing human leukocyte antigen molecules and costimulatory molecules; recruit leukocytes to the
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target site by expressing cytokines and chemokines. However, breach of tolerance in the biliary tree results in various cholangiopathies, exemplified by primary biliary
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cholangitis, primary sclerosing cholangitis and biliary atresia. Lessons learned from immune tolerance of the biliary tree will provide the basis for the development of
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effective therapeutic approaches against autoimmune biliary tract diseases.
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Introduction The human liver is a vital organ with distinctive anatomy, histology and heterogeneous cell populations. Although the physiological functions of the liver in digestion, storage of nutrients and detoxification have been studied for decades, it is not until recently that the liver is recognized as a lymphoid organ. Indeed, with persistent exposure to gut-derived
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dietary components, microbial products and environmental xenobiotics, the liver is armed with sophisticated immune mechanisms to modulate between a state of
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immunological tolerance and a state of responsiveness at any given time. This is of particular importance in maintaining immune homeostasis for immune tolerance to self
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and protection from foreign antigens within the liver microenvironments1, notably the biliary tree. In patients with autoimmune cholangitis, the biliary tree is the primary site of
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tolerance breakdown, chronic inflammation, immune attack and destruction of bile ducts.
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Anatomically, the biliary tree spreads over the entire liver and is an important integral component of the liver immunity. The biliary tree is composed of a complex network of interconnected ducts that increase in diameter from the canals of Hering to the choledochus2. The biliary system is composed of the intrahepatic bile ducts, which
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start at the canals of Hering and continue with bile ductules (< 15 μm), interlobular
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ducts (15–100 μm), area ducts (300–400 μm), septal ducts (100–300 μm), segmental ducts (400–800 μm), hepatic ducts (> 800 μm), and the extrahepatic bile ducts—that connect the liver and the pancreas with the intestine3. Long thought to be just a
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simple draining pipe for delivering bile from hepatocytes to the gallbladder and duodenum, the biliary tree is now regarded as a highly dynamic structure consisting
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of cells involved in bile secretion, bile acid reabsorption, drug metabolism and immune regulation4-7.
Both the intrahepatic and extrahepatic bile ducts are lined with biliary epithelial cells (BECs) named cholangiocytes, which are supported on a basement membrane and surrounded by connective tissue, extracellular matrix and the peribiliary plexus8. BECs display profound heterogeneity in morphological features, secretory function and responses to liver injury9-12. BECs function as a physical barrier and first line of defense against harsh environment from bile components. In normal conditions, 7
ACCEPTED MANUSCRIPT human bile is sterile. However, bacterial products such as lipopolysaccharide (LPS), lipoteichoic acid, bacterial DNA fragments and viral DNA sequences can be detected in bile samples of normal subjects and immune-compromised patients13, 14 15. Moreover, cultivable bacteria are also detectable in bile of patients with cholangiopathy16-18. Due to the exposure of the biliary tract to microbial components,
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chemical xenobiotics and foreign antigens, BECs need to respond to changes in their immediate microenvironment and have an active role in immune homeostasis
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through various immunologic pathways. In fact, a balance between inflammatory responses and immune tolerance is a key in mucosal environments.
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In this paper, we will discuss the current understanding on the role of cholangiocytes in maintaining immune tolerance in the biliary tree and the breach of tolerance in
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three autoimmune bile duct diseases, primary biliary cholangitis，primary sclerosing
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cholangitis and biliary atresia.
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How do cholangiocytes maintain immune tolerance of the biliary tree?
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As discussed earlier, the biliary tree is composed of a network of interconnected ducts of various diameters and is lined with BECs. Although the luminal surface of the bile duct is continually exposed to ever-changing components of bile such as
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microproducts, chemicals, no robust inflammatory response is elicited in BECs physiologically. Here, we will discuss how BECs tackle these challenges in
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maintaining immune tolerance (Figure 1).
1.Protection Against Pathogens via Pattern Recognition Receptors (PPRs). The luminal surface of BECs is continually exposed to Pathogen Associated Molecular Patterns (PAMPs) from bile and/or portal blood. Lipopolysaccharide (LPS) is one of the most abundant PAMPs derived from enteric bacteria. BEC handles LPS via “ endotoxin tolerance”, playing important roles in preventing endotoxin shock during infection and maintaining the homeostasis of organs such as the intestines with commensal bacterial flora and avoiding excessive tissue damage. Recent study 8
ACCEPTED MANUSCRIPT also suggest that variation in microbiome LPS is associated with innate immunity signaling and can be a contributing factor in autoimmunity19.
Toll like Receptors (TLRs) BECs recognize microbes and their constituents via cell surface receptors, known as
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pattern-recognition receptors (PPRs). TLRs are the best characterized epithelial PRRs recognizing PAMPs. Ten TLR members (TLR1 to TLR10) have been identified
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in humans20. At least TLR1-TLR5 have been reported in BECs13, 21-23. Additional evidence suggests that SV40-transformed human cholangiocytes could express
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mRNA from all ten TLRs24. Moreover, human and murine BECs also possess Tolllike receptor signaling related molecules. For example, TLR1-TLR5, myeloid
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differentiation factor 88 (MyD88), and IL-1 receptor-associated kinase-1 (IRAK-1) are distributed diffusely in the intrahepatic biliary tree in normal human livers21.
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Stimulation of BECs with PAMPs including Pam3CSK4 (TLR1/2 ligand), MALP-2 (TLR2/6 ligand), peptidoglycan (TLR2 ligand), and poly (I:C) (TLR3 ligand) induce the
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activation of TLRs downstream signaling in vitro, indicating that TLR expression in
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BECs is functional during bacterial, viral, and parasitic infections13, 21, 22. In response to LPS, TLR4 in conjunction with the TLR4 accessory proteins MD-2 and CD14 activates NF-κB through IL-1 signaling molecules, namely MyD88, IRAK-1, with the
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IRAK-M
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production of pro-inflammatory cytokines and antibiotics25.13
IRAK is a member of the TLR/IL-1R family of trans-membrane receptors. Four IRAKlike members have been identified: two active kinases, IRAK-1 and IRAK-4, and two inactive kinases, IRAK-2 and IRAK-M. Among them, IRAK-M negatively regulates TLR signaling by inhibiting the activation of MyD88 and IRAK-126. Recently, it is also reported that IRAK-M can inhibit TLR7-mediated production of cytokines and chemokines through interaction with IRAK-227. Under normal physiological condition, IRAK-M is diffusely distributed in intrahepatic biliary trees28. Stimulation of TLR-2 and TLR-4 in freshly isolated human intrahepatic BECs with bacterial PAMPs leads to the 9
ACCEPTED MANUSCRIPT up-regulation of IRAK-M and tolerant state in BECs29. This negative feedback mechanism of IRAK-M prevents BECs injury from excessive inflammatory responses. In addition to bacteria, Cryptosporidium parvum (C. parvum), a protozoan parasite causing intestinal and biliary diseases, may activate both TLR2 and TLR4 in cholangiocytes to initiate epithelial host responses and recruit these TLRs and ganglioside GM1 to membrane rafts30. In contrast to bacterial PAMPs, a TLR tolerance
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to a viral PAMP has not been detected in BECs31, even though IRAK-M mRNA
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expression is up-regulated by poly I:C stimulation. This is reasonable because dsRNA including viruses are recognized by TLR3, IFN-inducible helicase retinoic acid induced
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protein I (RIG-I), and melanoma differentiation associated gene-5 (MDA-5). The stimulation of these receptors by dsRNA transduces intracellular signals in a MyD88-
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independent way，that is, dsRNA-related immune response is not affected by IRAK-
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2. Production of Antimicrobial Biochemical Mediators
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As part of the host’s defenses against microbial infections, cholangiocytes produce
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proteins with antibacterial functions ( e.g. defensins, lactoferrin and lysozyme), antiviral functions (IFN-β1 and MxA) and other broad spectrum biomolecules such as
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cytokines and chemokines.
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Defensins are antimicrobial peptides with activity directed against a broad spectrum of microbes including bacteria and fungi. Defensins can be classified into two types, α- and β-defensins33. Six β-defensins (hBD1-6) have been identified in humans. Not all defensins are found in human bile. hBD1 is detected in bile and distributes throughout the intrahepatic biliary tree. hBD-1 is also constitutively expressed in cultured BECs. It is believed that hBD-1 is involved in the constitutive antimicrobial defense of the hepatobiliary system. hBD1 and hBD3 are also produced in cultured human BECs and SV40-transformed human cholangiocytes24, 34. In contrast, hBD2 is not expressed in normal livers. Expression of hBD-2 is induced in response to biliary 10
ACCEPTED MANUSCRIPT infections and may play a role in the localized antimicrobial defense. De novo production of hBD2 in bile ducts has been reported in patients with suppurative biliary inflammation such as biliary infections and hepatolithiasis and can be readily detected in their bile35. In cultured human BECs, activation of NF-κB by PAMPs such
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as LPS, E. coli, and C. parvum may also induce of hBD2 production24.
Cytokines and Chemokines
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In addition to antimicrobial peptides, cholangiocytes also produce various inflammatory cytokines and chemokines such as IL-8, IL-6, TNF-α, monocyte
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chemotactic protein-1(MCP-1), CX3CL1 and CXCL16, which are important chemoattractants for neutrophils, basophiles, monocytes, and T cells. IL-8
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expression is found in biliary epithelial cells from patients with cholangitis lenta, which is clinically characterized by bile ductular proliferation, ductular cholestasis, and
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ductular epithelial damage. Moreover IL-8 expression is closely associated with neutrophilic infiltration and reactive bile ductules36. IL-8 secreted by BECs is a
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potential target in the prevention of liver and biliary damage in diseased livers such
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as septic condition. IL-6 has been demonstrated to promote DNA synthesis in human biliary epithelial cells in vitro, indicating increased proliferative activity37. Interestingly，IL-6 and MCP-1 expression are also increased by TLR-4 activation
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without inflammatory cytokines. However, IL-8 expression is unaffected by TLR ligation34. Studies have shown that CX3CL1 (fractalkine) is detectable in BECs of
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small bile ducts in normal and diseased livers. Notably，CX3CL1 is increased significantly in impaired bile ducts of PBC patients38. In addition, the expression of CX3CL1 is elevated in serum concurrent with increased level of CX3CR1 in liver infiltrating mononuclear cells in PBC patients39. CX3CL1 has both chemoattractant and cell-adhesive functions and participates in the migration of leukocytes with its receptor CX3CR1 to target sites under physiological and pathological conditions. In PBC livers, the majority of mononuclear cells infiltrating around portal tracts are positive for CX3CR1, and most biliary intraepithelial lymphocytes in impaired bile ducts also express CX3CR138. It is suggested that production of CX3CL1 in BECs 11
ACCEPTED MANUSCRIPT leads to chemoattraction of CX3CR1-positive lymphocytes and mononuclear cells into portal tracts and biliary epithelia. Moreover, TLR3- activated BECs produce CX3CL1 after direct contact with TLR4-activated autologous monocytes40. Increased expression of CX3CL1 in the liver may be responsible for the development of biliary
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inflammation in PBC.
3. Secretion of Immunoglobulin A (IgA)
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The transport of IgA to the bile duct lumen is critical for mucosal immune defense in the biliary tract. Bile contains approximately twice the concentration of secretory IgA
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(SIgA) compared to upper intestinal fluid. SIgA is composed of two IgA joined together at their J chains and a secretory component. Polymeric immunoglobulin
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receptors on the surface of BECs are necessary for biliary transport of sIgA. Polymeric IgA binds to the secretory component on the basolateral side of BECs and
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is transported to the luminal surface, where the secretory component is cleaved and secreted along with the polymeric IgA41. Various studies have demonstrated that
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SIgA is involved in the protection of the biliary tract. For example, SIgA can bind to
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microorganisms, inhibit their motility, and prevent their adhesion to the mucosal membrane. Additionally, SIgA has been demonstrated to neutralize bacterial toxins42. Moreover, SIgA is able to prevent intracellular microbes from transiting through
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mucosal epithelium. SIgA and some other foreign antigen in the lamina propria may also be transported to the lumen through secretory component, and excrete the
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antigen to the mucosal43.
4. Presentation of Antigen One of the most interesting roles of BECs in the immune response is their potential ability to act as antigen presentation cells (APCs). APCs are characterized by expression of MHC class II and I, which are essential for antigen presentation to CD8+ and CD4+ T cells. In normal livers, HLA class I is expressed at a low frequency on BECs, while HLA class II molecules are not detected in biliary epithelium, which is critical for the maintenance of immune tolerance under 12
ACCEPTED MANUSCRIPT physiology condition. In vitro, cultured murine BECs constitutively express low levels of MHC Class I and MHC Class II molecules. However, upon cytomegalovirus (CMV) infection together with IFN-γ stimulation, HLA Class I and HLA Class II molecules were significantly augmented43. In contrast, in cultured human BECs, CMV-infection significantly enhanced the expression of HLA class I but not HLA class II molecules,
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reflecting the role of CD8+ T cells in viral responses44. Overexpression of HLA II has been demonstrated in damaged bile ducts from livers
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with allograft rejection, graft versus host disease(GVHD), PBC, and PSC45-47. To act as a competent APC for inducing primary immune responses and subsequent T cells
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activation, costimulatory molecules expression including CD40, CD80 (B7-1), and CD86(B7-2) on the surface of BECs are necessary. Various studies have been
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attempted to induce CD80 or CD86 expression in cultured normal BECs. However, no costimulation could be induced either in resting conditions or after stimulation with
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the proinflammatory cytokines, IFN-γ and TNF-α, or phorbol-12-myristate-13-acetate. Nevertheless, it is likely that BECs may still present antigen in an inefficient manner,
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as in B7-negative endothelial cells48. In fact, antigen presentation in the absence of
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CD80 may result in specific T-cell anergy, which exert suppressor function to inhibit subsequent T cell responses even in the presence of professional APCs49. In contrast, expression of CD86 was found in injured bile ducts of PBC and PSC
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patients46, 50. The expression of APC-related molecules on BECs suggests that BECs
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possess the capacity for antigen presentation.
5. Induction of Apoptosis in Leukocytes In patients of autoimmune liver diseases (AILD), cholangiocytes can express programmed-death (PD) ligands51, while PD receptors are expressed on leukocytes. Therefore, cholangiocytes are able to induce apoptosis in leukocytes through PDPDL ligation and limit the immune response52, 53. TNF-related apoptosis-inducing ligand (TRAIL) is expressed in the cholangiocytes of patients with PBC and PSC, but not in cholangiocytes from normal livers. Ligation of TRAIL receptors such as death receptors 4 and 5 (DR4-5) also result in apoptosis in leukocyte54. The expression of 13
ACCEPTED MANUSCRIPT apoptosis-related molecules on BECs is a likely mechanism in preventing excess immune responses55.
6. Transcriptional Regulation of Inflammatory Response Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor superfamily of ligand-activated transcription factors with anti-inflammatory effects56.
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When PPARγ is activated by its ligands including the prostaglandin D metabolite 15-
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deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and thiazolidinedione derivatives，the activation of NF-κB is attenuated and thereby the expression of proinflammatory
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cytokines such as TNF-αand IL-1β are significantly be inhibited57. In liver, PPARγ is constitutively expressed in cholangiocytes of intrahepatic bile ducts. PPARγ is
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suggested to be involved in biliary immune homeostasis by attenuating inflammatory signals in cholangiocytes to commensal PAMPs58. In cultured human BECs, it has
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been demonstrated that Th2 cytokine (IL-4) could up regulate PPARγ expression while Th1 cytokine (IFN-γ) could down regulate PPARγ expression. In patients with
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PBC, the expression of PPARγ is significantly decreased in the affected small bile
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ducts with Th1-dominant cytokine milieu, indicating an increased susceptibility to PAMPs. In cultured human BECs, 15d-PGJ2 treatment could inhibit PAMP (LPS or peptidoglycan)-induced NF-κB activation and TNF-α production58. Therefore, PPARγ
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ligands could likely provide protection against biliary inflammation in PBC.
Cholangiopathies Associated with Breach of Immune Tolerance Primary Biliary Cholangitis (PBC) PBC (formerly known as primary biliary cirrhosis) is a female predominant chronic autoimmune cholestatic liver disease characterized by the immune mediated selective destruction of interlobular bile ducts59. A key diagnostic element of PBC is the presence of antimitochondrial antibodies (AMA) against members of the 2-oxo acid dehydrogenase family, particularly the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2)60, 61. The breakdown immune tolerance to BECs 14
ACCEPTED MANUSCRIPT is believed to be the central culprit in the pathogenesis of PBC. In addition, molecular mimicry to xenobiotics and microbial proteins, genetic susceptibility and an imbalance in the immune microenvironment are also involved62-68. Inflammation and destruction of the intrahepatic bile duct is a characteristic histological and diagnostic feature of PBC, exemplified by infiltration of lymphocytes,
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macrophages and other inflammatory cells in the biliary epithelial layer69. Inflammatory responses, mediated by type 1 T helper cells, play critical roles in the
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loss of immunological tolerance to BEC, resulting in cholestasis and fibrosis70. In PBC, the expression of IFN-γ receptor on BECs, together with the Th1-dominant
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milieu (IFN-γ) upregulates the expression of TLRs, lead to increased susceptibility to PAMPs in the bile ducts, and impairs the regulation of biliary innate immunity. Recent
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studies demonstrate that apoptosis of cholangiocytes is a major event leading to immune mediated loss of bile ducts in PBC. Compared with normal controls,
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cholangiocytes from PBC patients show increased apoptosis as evidenced by increased DNA fragmentation71. In addition, cholangiocytes from PBC patients
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express significantly higher levels of Fas, FasL, TRAIL, perforin, and granzyme B54, 72
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. Compared with other chronic cholestatic diseases patients, cholangiocytes from
PBC patients have been demonstrated to present greater apoptosis with the up
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regulation of WAF1 and p5372-74.
In fact, cholangiocytes seem to be more than simply innocent victims of an immune
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attack, rather they may participate in the immune response by unique mechanisms75. BECs can translocate immunologically intact PDC-E2 to apoptotic bodies and create an apotope76. After phagocytizing and processing the apoptotic cholangiocytes, cholangiocytes are able to present novel mitochondrial self-peptides in conjunction with HLA class II acting as APCs77, 78. Thence autoreactive T cells against 2-OADC are recruited into the liver and gather around bile ducts79. Moreover, cholangiocytes are unique in secreting sIgA through transcytosis in the biliary lumen80 (Figure 2). The involvement of cholangiocytes in the pathogenesis of PBC is elegantly illustrated in an in vitro system where a combination of unique triad of BEC apotopes, 15
ACCEPTED MANUSCRIPT macrophages from patients with PBC, and AMAs lead to a significant proinflammatory cytokine production81.
Primary Sclerosing Cholangitis (PSC) PSC is a chronic bile duct disease characterized by fibrosis and dilatations of medium to large intrahepatic and extrahepatic bile ducts, leading to eventual biliary
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cirrhosis, failure or malignancy82. PSC is a challenging disease whose pathogenesis
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remains elusive. Concentric periductal fibrosis (onion skinning) with progression to stricturing of large bile ducts and obliteration of small bile ducts is the characteristic
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histological changes of PSC83. Detectable autoantibodies are found in as many as 97% of patients with PSC. The most commonly noted autoantibodies are
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antinuclear antibodies (ANA) and perinuclear anti-neutrophil cytoplasmic antibody (pANCA) which can be seen in 50% to 80% and 7% to 77% patients with
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PSC84.
Of note, apoptosis of BECs is speculated to be associated with the
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pathogenesis of PSC73. Histologically, increased expression of TRAIL has been
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found in BECs from PSC patients compared with healthy controls and patients with biliary stones54. However, Fas has been reported to be lower in the BECs of PSC compared to PBC. Moreover, TUNEL staining score was significantly lower in BECs
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from PSC patients than PBC patients85. Apoptosis of BECs may be of considerable
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importance for understanding pathogenic mechanisms in PSC.
Biliary Atresia
Biliary atresia is a severe infant biliary disease that destroys extrahepatic bile ducts and disrupts bile flow86, 87. The etiology of biliary atresia is poorly defined. Recent studies suggest that multiple factors are involved in the pathogenesis of disease, including: defects in embryogenesis, abnormal fetal or prenatal circulation, genetic factors, environmental toxins, viral infection, abnormal inflammation and autoimmunity88. A number of studies have provided insights on the mechanisms of epithelial injury in biliary atresia. 16
ACCEPTED MANUSCRIPT 1) Innate immune response against viruses: Rhesus rotavirus (RRV) can be detected in cholangiocytes of intrahepatic and extrahepatic bile ducts during infection89, 90. Furthermore, infection of BALB/c mice with reoviridae including type A RRV and type 3 reovirus (Abney) leads to cholestasis and biliary obstruction resembling human biliary atresia. 2) Enhanced apoptosis in cholangiocytes: Cholangiocytes are sensitive to TRAIL and
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Fas-mediated apoptosis91 . Enhanced cholangiocytes apoptosis is suggested as a
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mechanism in biliary atresia. In animal model of biliary atresia, blockade of caspase activity in vivo decreased the extent of injury to the biliary epithelium and supports
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the role of apoptosis in the pathogenesis of biliary atresia90. TLR3 is diffusely and constantly expressed in cholangiocytes of biliary atresia patients. There is elevated
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TRAIL and single-stranded DNA positive apoptosis in cholangiocytes with the activation of NF-κB and IRF-3 in patients with biliary atresia21. Furthermore,
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increased expression of TLR7, antimicrobial peptide hepcidin and MxA are also reported in early stage biliary atresia patients92. Altogether, these studies suggest
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that cholangiocytes can directly participate in antimicrobial innate immune response
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and induce apoptotic responses of infected cholangiocytes. 3) Epithelial mesenchymal transition (EMT) of cholangiocytes: Gradual decrease of epithelial markers (CK19 and E-cadherin), up regulated levels of the mesenchymal
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marker (S100A4) and EMT transcription factor (Snail), and increased susceptibility to transforming growth factor-β1 (TGF-β1) have been reported in cholangiocytes of
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patients with biliary atresia indicating the appearance of EMT in the biliary tract93.
Conclusion
The involvement of cholangiocytes in regulating the immune response has been widely assessed. Cholangiocytes are the first line of defense of the biliary tree against foreign substances, and are equipped to participate through various immunological pathways. Indeed, cholangiocytes protect against pathogens by TLRs-related signaling; maintain tolerance by expression of IRAK-M and PPARγ; limit immune response by inducing apoptosis of leukocytes; present antigen by 17
ACCEPTED MANUSCRIPT expressing human leukocyte antigen molecules and costimulatory molecules; recruit leukocytes to the target site by expressing cytokines and chemokines. However, in response to the immune attack, cholangiocytes may become active players in pathogenesis of PBC. Therefore, the regulatory activities of cholangiocytes are critical for the maintenance of immune tolerance in hepatic microenvironment.
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Lessons learned from immune tolerance of the biliary tree will enhance our understanding of immunobiology of cholangiocytes and provide the basis for the
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development of effective therapeutic approaches against autoimmune biliary tract diseases as well as the regulation of immune tolerance in other autoimmune
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diseases94, 95.
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Figure Legends Fig. 1. Schema for representative regulatory activities of BECs in maintaining of hepatic immune tolerance. BECs protect against pathogens by TLRs-related signaling; regulate inflammatory response by IRAK-M and PPARγ; recruit leukocytes to the target site by releasing cytokines and chemokines; induce apoptosis of
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leukocytes; present antigen by expressing human leukocyte antigen molecules.
Fig. 2. Schema for representative role of BECs in the pathology of PBC. In PBC,
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breach of tolerance results in a series of immune injuries to BECs. Cytotoxic T cells and Th1-dominant milieu induce apoptosis of BECs through FasL-Fas interactions
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and the secretion of perforin and granzyme B. BECs translocate intact PDC-E2 to apoptotic bodies and present novel mitochondrial self-peptides in conjunction with
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HLA class II acting as APCs. Thence autoreactive T cells are recruited into the liver and gather around bile ducts. In addition, cholangiocytes secret sIgA through
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transcytosis in the biliary lumen.
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Recognize pathogens Negative regulator of TLR signaling Anti-microbial, chemotactic Recruitment of immune cells
Protection of the biliary tract against infection
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Antigen presentation
Induction of apoptosis in leukocytes Negative transcriptional regulator of inflammatory response
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Human leukocyte antigen molecules and costimulatory molecules PDL, TRAIL, DR4, DR5 PPARγ
Function
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BECs expression TLRs IRAK-M Defensins Cytokines and chemokines (IL-8, IL-6, TNF-α, MCP-1, CX3CL1 and CXCL16) SIgA
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Haiyan Zhang, Patrick S.C. Leung, M. Eric Gershwin, Xiong Ma PII: DOI: Reference:
S0925-4439(17)30301-0 doi: 10.1016/j.bbadis.2017.08.019 BBADIS 64869
To appear in: Received date: Revised date: Accepted date:
15 May 2017 3 August 2017 9 August 2017
Please cite this article as: Haiyan Zhang, Patrick S.C. Leung, M. Eric Gershwin, Xiong Ma , How the biliary tree maintains immune tolerance?, (2017), doi: 10.1016/ j.bbadis.2017.08.019
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How the biliary tree maintains immune tolerance?
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Haiyan Zhang1, Patrick S.C.Leung2, M. Eric Gershwin2 and Xiong Ma1
Division
of
Gastroenterology and
Hepatology,
Key Laboratory of
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Affiliations
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Gastroenterology and Hepatology, Ministry of Health, State Key Laboratory for Oncogenes and Related Genes, Renji Hospital, School of Medicine, Shanghai
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Jiao Tong University; Shanghai Institute of Digestive Disease; 145 Middle Shandong Road, Shanghai 200001, China Division of Rheumatology, Allergy, and Clinical Immunology, University of
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California at Davis, Davis, California, USA
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Correspondence:
Xiong Ma, MD, Ph.D. Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Institute of Digestive Disease, 145 Middle Shandong road, Shanghai 200001, China. Email: [email protected];
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ACCEPTED MANUSCRIPT Keywords: Cholangiocytes; biliary tree; immune tolerance; primary biliary cholangitis; primary sclerosing cholangitis; biliary atresia
Conflict of interest The authors declare no financial conflict of interest exists
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Financial Support
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This work was supported by awards from the National Natural Science
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NU
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Foundation of China (#81325002 and 81620108002 to Xiong Ma)
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ACCEPTED MANUSCRIPT Abbreviations:
Dendritic Cells
NKT cells
Natural Killer T cells
BECs
Biliary Epithelial Cells
LPS
Lipopolysaccharide
PAMPs
Pathogen Associated Molecular Patterns
PRRs
Pattern-Recognition Receptors
TLRs
Toll-Like Receptors
SV40
Simian Virus 40
MyD88
Myeloid Differentiation Factor 88
IRAK-1
IL-1 Receptor-Associated Kinase-1
IFN-γ
Interferon- γ
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DCs
RIG-1
Retinoic acid Induced Protein 1 Melanoma Differentiation Gene-5 Human β-defensins
hBD
Monocyte Chemotatctic Protein-1
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MCP-1
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CX3CL1 CX3CR1 SIgA
PSC AILD PD
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PBC
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CMV GVHD
Associated
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MDA-5
Fractalkine CX3CL1 Receptor Secretory IgA Cytomegalovirus Graft Versus Host Disease Primary Biliary Cholangitis Primary Sclerosing Cholangitis Autoimmune Liver Diseases Programmed-death
TRAIL
TNF-related Apoptosis-inducing Ligand
PPARγ RRV
Peroxisome Receptor γ Rhesus Rotavirus
EMT
Epithelial-Mesenchymal Transition
ANA
Antinuclear antibodies
3
Proliferator-activated
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Perinuclear anti-neutrophil cytoplasmic
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antibody
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ACCEPTED MANUSCRIPT Research Highlights 1. BECs are the first line of defense of the biliary tree against foreign substances. 2. BECs play important roles in maintaining tolerance through various immunological pathways.
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3. Breach of tolerance in the biliary tree results in various cholangiopathies.
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Abstract The liver is a vital organ with distinctive anatomy, histology and heterogeneous cell populations. These characteristics are of particular importance in maintaining immune homeostasis within the liver microenvironments, notably the biliary tree. Cholangiocytes are the first line of defense of the biliary tree against foreign
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substances, and are equipped to participate through various immunological pathways. Indeed, cholangiocytes protect against pathogens by TLRs-related
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signaling; maintain tolerance by expression of IRAK-M and PPARγ; limit immune
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response by inducing apoptosis of leukocytes; present antigen by expressing human leukocyte antigen molecules and costimulatory molecules; recruit leukocytes to the
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target site by expressing cytokines and chemokines. However, breach of tolerance in the biliary tree results in various cholangiopathies, exemplified by primary biliary
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cholangitis, primary sclerosing cholangitis and biliary atresia. Lessons learned from immune tolerance of the biliary tree will provide the basis for the development of
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effective therapeutic approaches against autoimmune biliary tract diseases.
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Introduction The human liver is a vital organ with distinctive anatomy, histology and heterogeneous cell populations. Although the physiological functions of the liver in digestion, storage of nutrients and detoxification have been studied for decades, it is not until recently that the liver is recognized as a lymphoid organ. Indeed, with persistent exposure to gut-derived
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dietary components, microbial products and environmental xenobiotics, the liver is armed with sophisticated immune mechanisms to modulate between a state of
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immunological tolerance and a state of responsiveness at any given time. This is of particular importance in maintaining immune homeostasis for immune tolerance to self
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and protection from foreign antigens within the liver microenvironments1, notably the biliary tree. In patients with autoimmune cholangitis, the biliary tree is the primary site of
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tolerance breakdown, chronic inflammation, immune attack and destruction of bile ducts.
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Anatomically, the biliary tree spreads over the entire liver and is an important integral component of the liver immunity. The biliary tree is composed of a complex network of interconnected ducts that increase in diameter from the canals of Hering to the choledochus2. The biliary system is composed of the intrahepatic bile ducts, which
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start at the canals of Hering and continue with bile ductules (< 15 μm), interlobular
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ducts (15–100 μm), area ducts (300–400 μm), septal ducts (100–300 μm), segmental ducts (400–800 μm), hepatic ducts (> 800 μm), and the extrahepatic bile ducts—that connect the liver and the pancreas with the intestine3. Long thought to be just a
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simple draining pipe for delivering bile from hepatocytes to the gallbladder and duodenum, the biliary tree is now regarded as a highly dynamic structure consisting
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of cells involved in bile secretion, bile acid reabsorption, drug metabolism and immune regulation4-7.
Both the intrahepatic and extrahepatic bile ducts are lined with biliary epithelial cells (BECs) named cholangiocytes, which are supported on a basement membrane and surrounded by connective tissue, extracellular matrix and the peribiliary plexus8. BECs display profound heterogeneity in morphological features, secretory function and responses to liver injury9-12. BECs function as a physical barrier and first line of defense against harsh environment from bile components. In normal conditions, 7
ACCEPTED MANUSCRIPT human bile is sterile. However, bacterial products such as lipopolysaccharide (LPS), lipoteichoic acid, bacterial DNA fragments and viral DNA sequences can be detected in bile samples of normal subjects and immune-compromised patients13, 14 15. Moreover, cultivable bacteria are also detectable in bile of patients with cholangiopathy16-18. Due to the exposure of the biliary tract to microbial components,
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chemical xenobiotics and foreign antigens, BECs need to respond to changes in their immediate microenvironment and have an active role in immune homeostasis
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through various immunologic pathways. In fact, a balance between inflammatory responses and immune tolerance is a key in mucosal environments.
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In this paper, we will discuss the current understanding on the role of cholangiocytes in maintaining immune tolerance in the biliary tree and the breach of tolerance in
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three autoimmune bile duct diseases, primary biliary cholangitis，primary sclerosing
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cholangitis and biliary atresia.
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How do cholangiocytes maintain immune tolerance of the biliary tree?
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As discussed earlier, the biliary tree is composed of a network of interconnected ducts of various diameters and is lined with BECs. Although the luminal surface of the bile duct is continually exposed to ever-changing components of bile such as
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microproducts, chemicals, no robust inflammatory response is elicited in BECs physiologically. Here, we will discuss how BECs tackle these challenges in
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maintaining immune tolerance (Figure 1).
1.Protection Against Pathogens via Pattern Recognition Receptors (PPRs). The luminal surface of BECs is continually exposed to Pathogen Associated Molecular Patterns (PAMPs) from bile and/or portal blood. Lipopolysaccharide (LPS) is one of the most abundant PAMPs derived from enteric bacteria. BEC handles LPS via “ endotoxin tolerance”, playing important roles in preventing endotoxin shock during infection and maintaining the homeostasis of organs such as the intestines with commensal bacterial flora and avoiding excessive tissue damage. Recent study 8
ACCEPTED MANUSCRIPT also suggest that variation in microbiome LPS is associated with innate immunity signaling and can be a contributing factor in autoimmunity19.
Toll like Receptors (TLRs) BECs recognize microbes and their constituents via cell surface receptors, known as
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pattern-recognition receptors (PPRs). TLRs are the best characterized epithelial PRRs recognizing PAMPs. Ten TLR members (TLR1 to TLR10) have been identified
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in humans20. At least TLR1-TLR5 have been reported in BECs13, 21-23. Additional evidence suggests that SV40-transformed human cholangiocytes could express
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mRNA from all ten TLRs24. Moreover, human and murine BECs also possess Tolllike receptor signaling related molecules. For example, TLR1-TLR5, myeloid
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differentiation factor 88 (MyD88), and IL-1 receptor-associated kinase-1 (IRAK-1) are distributed diffusely in the intrahepatic biliary tree in normal human livers21.
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Stimulation of BECs with PAMPs including Pam3CSK4 (TLR1/2 ligand), MALP-2 (TLR2/6 ligand), peptidoglycan (TLR2 ligand), and poly (I:C) (TLR3 ligand) induce the
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activation of TLRs downstream signaling in vitro, indicating that TLR expression in
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BECs is functional during bacterial, viral, and parasitic infections13, 21, 22. In response to LPS, TLR4 in conjunction with the TLR4 accessory proteins MD-2 and CD14 activates NF-κB through IL-1 signaling molecules, namely MyD88, IRAK-1, with the
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IRAK-M
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production of pro-inflammatory cytokines and antibiotics25.13
IRAK is a member of the TLR/IL-1R family of trans-membrane receptors. Four IRAKlike members have been identified: two active kinases, IRAK-1 and IRAK-4, and two inactive kinases, IRAK-2 and IRAK-M. Among them, IRAK-M negatively regulates TLR signaling by inhibiting the activation of MyD88 and IRAK-126. Recently, it is also reported that IRAK-M can inhibit TLR7-mediated production of cytokines and chemokines through interaction with IRAK-227. Under normal physiological condition, IRAK-M is diffusely distributed in intrahepatic biliary trees28. Stimulation of TLR-2 and TLR-4 in freshly isolated human intrahepatic BECs with bacterial PAMPs leads to the 9
ACCEPTED MANUSCRIPT up-regulation of IRAK-M and tolerant state in BECs29. This negative feedback mechanism of IRAK-M prevents BECs injury from excessive inflammatory responses. In addition to bacteria, Cryptosporidium parvum (C. parvum), a protozoan parasite causing intestinal and biliary diseases, may activate both TLR2 and TLR4 in cholangiocytes to initiate epithelial host responses and recruit these TLRs and ganglioside GM1 to membrane rafts30. In contrast to bacterial PAMPs, a TLR tolerance
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to a viral PAMP has not been detected in BECs31, even though IRAK-M mRNA
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expression is up-regulated by poly I:C stimulation. This is reasonable because dsRNA including viruses are recognized by TLR3, IFN-inducible helicase retinoic acid induced
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protein I (RIG-I), and melanoma differentiation associated gene-5 (MDA-5). The stimulation of these receptors by dsRNA transduces intracellular signals in a MyD88-
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independent way，that is, dsRNA-related immune response is not affected by IRAK-
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M32.
2. Production of Antimicrobial Biochemical Mediators
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As part of the host’s defenses against microbial infections, cholangiocytes produce
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proteins with antibacterial functions ( e.g. defensins, lactoferrin and lysozyme), antiviral functions (IFN-β1 and MxA) and other broad spectrum biomolecules such as
Defensins
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cytokines and chemokines.
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Defensins are antimicrobial peptides with activity directed against a broad spectrum of microbes including bacteria and fungi. Defensins can be classified into two types, α- and β-defensins33. Six β-defensins (hBD1-6) have been identified in humans. Not all defensins are found in human bile. hBD1 is detected in bile and distributes throughout the intrahepatic biliary tree. hBD-1 is also constitutively expressed in cultured BECs. It is believed that hBD-1 is involved in the constitutive antimicrobial defense of the hepatobiliary system. hBD1 and hBD3 are also produced in cultured human BECs and SV40-transformed human cholangiocytes24, 34. In contrast, hBD2 is not expressed in normal livers. Expression of hBD-2 is induced in response to biliary 10
ACCEPTED MANUSCRIPT infections and may play a role in the localized antimicrobial defense. De novo production of hBD2 in bile ducts has been reported in patients with suppurative biliary inflammation such as biliary infections and hepatolithiasis and can be readily detected in their bile35. In cultured human BECs, activation of NF-κB by PAMPs such
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as LPS, E. coli, and C. parvum may also induce of hBD2 production24.
Cytokines and Chemokines
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In addition to antimicrobial peptides, cholangiocytes also produce various inflammatory cytokines and chemokines such as IL-8, IL-6, TNF-α, monocyte
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chemotactic protein-1(MCP-1), CX3CL1 and CXCL16, which are important chemoattractants for neutrophils, basophiles, monocytes, and T cells. IL-8
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expression is found in biliary epithelial cells from patients with cholangitis lenta, which is clinically characterized by bile ductular proliferation, ductular cholestasis, and
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ductular epithelial damage. Moreover IL-8 expression is closely associated with neutrophilic infiltration and reactive bile ductules36. IL-8 secreted by BECs is a
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potential target in the prevention of liver and biliary damage in diseased livers such
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as septic condition. IL-6 has been demonstrated to promote DNA synthesis in human biliary epithelial cells in vitro, indicating increased proliferative activity37. Interestingly，IL-6 and MCP-1 expression are also increased by TLR-4 activation
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without inflammatory cytokines. However, IL-8 expression is unaffected by TLR ligation34. Studies have shown that CX3CL1 (fractalkine) is detectable in BECs of
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small bile ducts in normal and diseased livers. Notably，CX3CL1 is increased significantly in impaired bile ducts of PBC patients38. In addition, the expression of CX3CL1 is elevated in serum concurrent with increased level of CX3CR1 in liver infiltrating mononuclear cells in PBC patients39. CX3CL1 has both chemoattractant and cell-adhesive functions and participates in the migration of leukocytes with its receptor CX3CR1 to target sites under physiological and pathological conditions. In PBC livers, the majority of mononuclear cells infiltrating around portal tracts are positive for CX3CR1, and most biliary intraepithelial lymphocytes in impaired bile ducts also express CX3CR138. It is suggested that production of CX3CL1 in BECs 11
ACCEPTED MANUSCRIPT leads to chemoattraction of CX3CR1-positive lymphocytes and mononuclear cells into portal tracts and biliary epithelia. Moreover, TLR3- activated BECs produce CX3CL1 after direct contact with TLR4-activated autologous monocytes40. Increased expression of CX3CL1 in the liver may be responsible for the development of biliary
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inflammation in PBC.
3. Secretion of Immunoglobulin A (IgA)
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The transport of IgA to the bile duct lumen is critical for mucosal immune defense in the biliary tract. Bile contains approximately twice the concentration of secretory IgA
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(SIgA) compared to upper intestinal fluid. SIgA is composed of two IgA joined together at their J chains and a secretory component. Polymeric immunoglobulin
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receptors on the surface of BECs are necessary for biliary transport of sIgA. Polymeric IgA binds to the secretory component on the basolateral side of BECs and
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is transported to the luminal surface, where the secretory component is cleaved and secreted along with the polymeric IgA41. Various studies have demonstrated that
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SIgA is involved in the protection of the biliary tract. For example, SIgA can bind to
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microorganisms, inhibit their motility, and prevent their adhesion to the mucosal membrane. Additionally, SIgA has been demonstrated to neutralize bacterial toxins42. Moreover, SIgA is able to prevent intracellular microbes from transiting through
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mucosal epithelium. SIgA and some other foreign antigen in the lamina propria may also be transported to the lumen through secretory component, and excrete the
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antigen to the mucosal43.
4. Presentation of Antigen One of the most interesting roles of BECs in the immune response is their potential ability to act as antigen presentation cells (APCs). APCs are characterized by expression of MHC class II and I, which are essential for antigen presentation to CD8+ and CD4+ T cells. In normal livers, HLA class I is expressed at a low frequency on BECs, while HLA class II molecules are not detected in biliary epithelium, which is critical for the maintenance of immune tolerance under 12
ACCEPTED MANUSCRIPT physiology condition. In vitro, cultured murine BECs constitutively express low levels of MHC Class I and MHC Class II molecules. However, upon cytomegalovirus (CMV) infection together with IFN-γ stimulation, HLA Class I and HLA Class II molecules were significantly augmented43. In contrast, in cultured human BECs, CMV-infection significantly enhanced the expression of HLA class I but not HLA class II molecules,
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reflecting the role of CD8+ T cells in viral responses44. Overexpression of HLA II has been demonstrated in damaged bile ducts from livers
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with allograft rejection, graft versus host disease(GVHD), PBC, and PSC45-47. To act as a competent APC for inducing primary immune responses and subsequent T cells
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activation, costimulatory molecules expression including CD40, CD80 (B7-1), and CD86(B7-2) on the surface of BECs are necessary. Various studies have been
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attempted to induce CD80 or CD86 expression in cultured normal BECs. However, no costimulation could be induced either in resting conditions or after stimulation with
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the proinflammatory cytokines, IFN-γ and TNF-α, or phorbol-12-myristate-13-acetate. Nevertheless, it is likely that BECs may still present antigen in an inefficient manner,
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as in B7-negative endothelial cells48. In fact, antigen presentation in the absence of
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CD80 may result in specific T-cell anergy, which exert suppressor function to inhibit subsequent T cell responses even in the presence of professional APCs49. In contrast, expression of CD86 was found in injured bile ducts of PBC and PSC
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patients46, 50. The expression of APC-related molecules on BECs suggests that BECs
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possess the capacity for antigen presentation.
5. Induction of Apoptosis in Leukocytes In patients of autoimmune liver diseases (AILD), cholangiocytes can express programmed-death (PD) ligands51, while PD receptors are expressed on leukocytes. Therefore, cholangiocytes are able to induce apoptosis in leukocytes through PDPDL ligation and limit the immune response52, 53. TNF-related apoptosis-inducing ligand (TRAIL) is expressed in the cholangiocytes of patients with PBC and PSC, but not in cholangiocytes from normal livers. Ligation of TRAIL receptors such as death receptors 4 and 5 (DR4-5) also result in apoptosis in leukocyte54. The expression of 13
ACCEPTED MANUSCRIPT apoptosis-related molecules on BECs is a likely mechanism in preventing excess immune responses55.
6. Transcriptional Regulation of Inflammatory Response Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor superfamily of ligand-activated transcription factors with anti-inflammatory effects56.
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When PPARγ is activated by its ligands including the prostaglandin D metabolite 15-
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deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and thiazolidinedione derivatives，the activation of NF-κB is attenuated and thereby the expression of proinflammatory
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cytokines such as TNF-αand IL-1β are significantly be inhibited57. In liver, PPARγ is constitutively expressed in cholangiocytes of intrahepatic bile ducts. PPARγ is
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suggested to be involved in biliary immune homeostasis by attenuating inflammatory signals in cholangiocytes to commensal PAMPs58. In cultured human BECs, it has
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been demonstrated that Th2 cytokine (IL-4) could up regulate PPARγ expression while Th1 cytokine (IFN-γ) could down regulate PPARγ expression. In patients with
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PBC, the expression of PPARγ is significantly decreased in the affected small bile
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ducts with Th1-dominant cytokine milieu, indicating an increased susceptibility to PAMPs. In cultured human BECs, 15d-PGJ2 treatment could inhibit PAMP (LPS or peptidoglycan)-induced NF-κB activation and TNF-α production58. Therefore, PPARγ
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ligands could likely provide protection against biliary inflammation in PBC.
Cholangiopathies Associated with Breach of Immune Tolerance Primary Biliary Cholangitis (PBC) PBC (formerly known as primary biliary cirrhosis) is a female predominant chronic autoimmune cholestatic liver disease characterized by the immune mediated selective destruction of interlobular bile ducts59. A key diagnostic element of PBC is the presence of antimitochondrial antibodies (AMA) against members of the 2-oxo acid dehydrogenase family, particularly the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2)60, 61. The breakdown immune tolerance to BECs 14
ACCEPTED MANUSCRIPT is believed to be the central culprit in the pathogenesis of PBC. In addition, molecular mimicry to xenobiotics and microbial proteins, genetic susceptibility and an imbalance in the immune microenvironment are also involved62-68. Inflammation and destruction of the intrahepatic bile duct is a characteristic histological and diagnostic feature of PBC, exemplified by infiltration of lymphocytes,
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macrophages and other inflammatory cells in the biliary epithelial layer69. Inflammatory responses, mediated by type 1 T helper cells, play critical roles in the
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loss of immunological tolerance to BEC, resulting in cholestasis and fibrosis70. In PBC, the expression of IFN-γ receptor on BECs, together with the Th1-dominant
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milieu (IFN-γ) upregulates the expression of TLRs, lead to increased susceptibility to PAMPs in the bile ducts, and impairs the regulation of biliary innate immunity. Recent
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studies demonstrate that apoptosis of cholangiocytes is a major event leading to immune mediated loss of bile ducts in PBC. Compared with normal controls,
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cholangiocytes from PBC patients show increased apoptosis as evidenced by increased DNA fragmentation71. In addition, cholangiocytes from PBC patients
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express significantly higher levels of Fas, FasL, TRAIL, perforin, and granzyme B54, 72
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. Compared with other chronic cholestatic diseases patients, cholangiocytes from
PBC patients have been demonstrated to present greater apoptosis with the up
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regulation of WAF1 and p5372-74.
In fact, cholangiocytes seem to be more than simply innocent victims of an immune
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attack, rather they may participate in the immune response by unique mechanisms75. BECs can translocate immunologically intact PDC-E2 to apoptotic bodies and create an apotope76. After phagocytizing and processing the apoptotic cholangiocytes, cholangiocytes are able to present novel mitochondrial self-peptides in conjunction with HLA class II acting as APCs77, 78. Thence autoreactive T cells against 2-OADC are recruited into the liver and gather around bile ducts79. Moreover, cholangiocytes are unique in secreting sIgA through transcytosis in the biliary lumen80 (Figure 2). The involvement of cholangiocytes in the pathogenesis of PBC is elegantly illustrated in an in vitro system where a combination of unique triad of BEC apotopes, 15
ACCEPTED MANUSCRIPT macrophages from patients with PBC, and AMAs lead to a significant proinflammatory cytokine production81.
Primary Sclerosing Cholangitis (PSC) PSC is a chronic bile duct disease characterized by fibrosis and dilatations of medium to large intrahepatic and extrahepatic bile ducts, leading to eventual biliary
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cirrhosis, failure or malignancy82. PSC is a challenging disease whose pathogenesis
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remains elusive. Concentric periductal fibrosis (onion skinning) with progression to stricturing of large bile ducts and obliteration of small bile ducts is the characteristic
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histological changes of PSC83. Detectable autoantibodies are found in as many as 97% of patients with PSC. The most commonly noted autoantibodies are
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antinuclear antibodies (ANA) and perinuclear anti-neutrophil cytoplasmic antibody (pANCA) which can be seen in 50% to 80% and 7% to 77% patients with
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PSC84.
Of note, apoptosis of BECs is speculated to be associated with the
D
pathogenesis of PSC73. Histologically, increased expression of TRAIL has been
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found in BECs from PSC patients compared with healthy controls and patients with biliary stones54. However, Fas has been reported to be lower in the BECs of PSC compared to PBC. Moreover, TUNEL staining score was significantly lower in BECs
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from PSC patients than PBC patients85. Apoptosis of BECs may be of considerable
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importance for understanding pathogenic mechanisms in PSC.
Biliary Atresia
Biliary atresia is a severe infant biliary disease that destroys extrahepatic bile ducts and disrupts bile flow86, 87. The etiology of biliary atresia is poorly defined. Recent studies suggest that multiple factors are involved in the pathogenesis of disease, including: defects in embryogenesis, abnormal fetal or prenatal circulation, genetic factors, environmental toxins, viral infection, abnormal inflammation and autoimmunity88. A number of studies have provided insights on the mechanisms of epithelial injury in biliary atresia. 16
ACCEPTED MANUSCRIPT 1) Innate immune response against viruses: Rhesus rotavirus (RRV) can be detected in cholangiocytes of intrahepatic and extrahepatic bile ducts during infection89, 90. Furthermore, infection of BALB/c mice with reoviridae including type A RRV and type 3 reovirus (Abney) leads to cholestasis and biliary obstruction resembling human biliary atresia. 2) Enhanced apoptosis in cholangiocytes: Cholangiocytes are sensitive to TRAIL and
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Fas-mediated apoptosis91 . Enhanced cholangiocytes apoptosis is suggested as a
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mechanism in biliary atresia. In animal model of biliary atresia, blockade of caspase activity in vivo decreased the extent of injury to the biliary epithelium and supports
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the role of apoptosis in the pathogenesis of biliary atresia90. TLR3 is diffusely and constantly expressed in cholangiocytes of biliary atresia patients. There is elevated
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TRAIL and single-stranded DNA positive apoptosis in cholangiocytes with the activation of NF-κB and IRF-3 in patients with biliary atresia21. Furthermore,
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increased expression of TLR7, antimicrobial peptide hepcidin and MxA are also reported in early stage biliary atresia patients92. Altogether, these studies suggest
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that cholangiocytes can directly participate in antimicrobial innate immune response
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and induce apoptotic responses of infected cholangiocytes. 3) Epithelial mesenchymal transition (EMT) of cholangiocytes: Gradual decrease of epithelial markers (CK19 and E-cadherin), up regulated levels of the mesenchymal
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marker (S100A4) and EMT transcription factor (Snail), and increased susceptibility to transforming growth factor-β1 (TGF-β1) have been reported in cholangiocytes of
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patients with biliary atresia indicating the appearance of EMT in the biliary tract93.
Conclusion
The involvement of cholangiocytes in regulating the immune response has been widely assessed. Cholangiocytes are the first line of defense of the biliary tree against foreign substances, and are equipped to participate through various immunological pathways. Indeed, cholangiocytes protect against pathogens by TLRs-related signaling; maintain tolerance by expression of IRAK-M and PPARγ; limit immune response by inducing apoptosis of leukocytes; present antigen by 17
ACCEPTED MANUSCRIPT expressing human leukocyte antigen molecules and costimulatory molecules; recruit leukocytes to the target site by expressing cytokines and chemokines. However, in response to the immune attack, cholangiocytes may become active players in pathogenesis of PBC. Therefore, the regulatory activities of cholangiocytes are critical for the maintenance of immune tolerance in hepatic microenvironment.
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Lessons learned from immune tolerance of the biliary tree will enhance our understanding of immunobiology of cholangiocytes and provide the basis for the
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development of effective therapeutic approaches against autoimmune biliary tract diseases as well as the regulation of immune tolerance in other autoimmune
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diseases94, 95.
18
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Figure Legends Fig. 1. Schema for representative regulatory activities of BECs in maintaining of hepatic immune tolerance. BECs protect against pathogens by TLRs-related signaling; regulate inflammatory response by IRAK-M and PPARγ; recruit leukocytes to the target site by releasing cytokines and chemokines; induce apoptosis of
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leukocytes; present antigen by expressing human leukocyte antigen molecules.
Fig. 2. Schema for representative role of BECs in the pathology of PBC. In PBC,
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breach of tolerance results in a series of immune injuries to BECs. Cytotoxic T cells and Th1-dominant milieu induce apoptosis of BECs through FasL-Fas interactions
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and the secretion of perforin and granzyme B. BECs translocate intact PDC-E2 to apoptotic bodies and present novel mitochondrial self-peptides in conjunction with
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HLA class II acting as APCs. Thence autoreactive T cells are recruited into the liver and gather around bile ducts. In addition, cholangiocytes secret sIgA through
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ACCEPTED MANUSCRIPT Table 1. The immunophysiology of BECs in maintaining immune tolerance
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Recognize pathogens Negative regulator of TLR signaling Anti-microbial, chemotactic Recruitment of immune cells
Protection of the biliary tract against infection
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Antigen presentation
Induction of apoptosis in leukocytes Negative transcriptional regulator of inflammatory response
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Human leukocyte antigen molecules and costimulatory molecules PDL, TRAIL, DR4, DR5 PPARγ
Function
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BECs expression TLRs IRAK-M Defensins Cytokines and chemokines (IL-8, IL-6, TNF-α, MCP-1, CX3CL1 and CXCL16) SIgA
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