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Objective: Primary biliary cholangitis (PBC) is a chronic progressive cholestatic liver disease. In recent years, researchers have found that cysteine-rich angiogenic inducer 61 (Cyr61, also known as CCN1) has a potential role in reducing portal inflammation in patients with PBC. This study aimed to explore the relationship between Cyr61 and PBC to provide new ideas and an experimental basis for the clinical treatment of PBC., Methods: After induction of the overexpression of Cyr61 in a mouse model of PBC using recombinant adenovirus, hematoxylin and eosin staining and pathological scores were used to indicate intrahepatic inflammation and bile duct damage. Real-time PCR was used to detect changes in inflammation-related cytokines in the liver. To further study the mechanism, we assessed whether Cyr61 protects bile duct epithelial cells from cytotoxic effects., Results: Serum and hepatic Cyr61 levels were increased in the murine model of PBC. Overexpression of Cyr61 alleviated hepatic inflammation and bile duct injury in vivo. Cyr61 inhibited the cytotoxic effects of CD8 + T cells by acting on biliary epithelial cells (BECs) in vitro., Conclusion: Our results provide novel insight into the pathogenesis of PBC and suggest that Cyr61 plays a dominant role in the cytotoxic effects on BECs in PBC. Consequently, therapeutic strategies targeting Cyr61 could be a potent therapy for PBC., (© 2021. Huazhong University of Science and Technology.)

Background and Aims: Cholestasis is characterized by increased total bile acid (TBA) levels, which are regulated by farnesoid X receptor (FXR)/FGF15. Patients with primary sclerosing cholangitis (PSC) typically present with inflammatory bowel disease (IBD). Mast cells (MCs) (i) express FXR and (ii) infiltrate the liver during cholestasis promoting liver fibrosis. In bile-duct-ligated (BDL) MC-deficient mice (B6.Cg-Kit W-sh /HNihrJaeBsmJ [Kit W-sh ]), ductular reaction (DR) and liver fibrosis decrease compared with BDL wild type, and MC injection exacerbates liver damage in normal mice., Approach and Results: In this study, we demonstrated that MC-FXR regulates biliary FXR/FGF15, DR, and hepatic fibrosis and alters intestinal FXR/FGF15. We found increased MC number and biliary FXR expression in patients with liver injury compared with control. Histamine and FGF19 serum levels and small heterodimer partner expression increase in patients PSC and PSC-IBD compared with healthy controls. MC injection increased liver damage, DR, inflammation, biliary senescence/senescence-associated secretory phenotype (SASP), fibrosis, and histamine in Kit W-sh mice. Inhibition of MC-FXR before injection reduced these parameters. BDL and Kit W-sh mice injected with MCs displayed increased TBA content, biliary FXR/FGF15, and intestinal inflammation, which decreased in BDL Kit W-sh and Kit W-sh mice injected with MC-FXR. MCs increased ileal FXR/FGF15 expression in Kit W-sh mice that was reduced following FXR inhibition. BDL and multidrug resistance 2/ATP-binding cassette family 2 member 4 knockout (Mdr2 -/- ) mice, models of PSC, displayed increased intestinal MC infiltration and FXR/FGF15 expression. These were reduced following MC stabilization with cromolyn sodium in Mdr2 -/- mice. In vitro, MC-FXR inhibition decreased biliary proliferation/SASP/FGF and hepatic stellate cell activation., Conclusions: Our studies demonstrate that MC-FXR plays a key role in liver damage and DR, including TBA regulation through alteration of intestinal and biliary FXR/FGF15 signaling., (© 2021 Wiley Periodicals LLC. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

Introduction: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 virus, is a predominantly respiratory tract infection with the capacity to affect multiple organ systems. Abnormal liver tests, mainly transaminase elevations, have been reported in hospitalized patients. We describe a syndrome of cholangiopathy in patients recovering from severe COVID-19 characterized by marked elevation in serum alkaline phosphatase (ALP) accompanied by evidence of bile duct injury on imaging., Methods: We conducted a retrospective study of COVID-19 patients admitted to our institution from March 1, 2020, to August 15, 2020, on whom the hepatology service was consulted for abnormal liver tests. Bile duct injury was identified by abnormal liver tests with serum ALP > 3x upper limit of normal and abnormal findings on magnetic resonance cholangiopacreatography. Clinical, laboratory, radiological, and histological findings were recorded in a Research Electronic Data Capture database., Results: Twelve patients were identified, 11 men and 1 woman, with a mean age of 58 years. Mean time from COVID-19 diagnosis to diagnosis of cholangiopathy was 118 days. Peak median serum alanine aminotransferase was 661 U/L and peak median serum ALP was 1855 U/L. Marked elevations of erythrocyte sedimentation rate, C-reactive protein, and D-dimers were common. Magnetic resonance cholangiopacreatography findings included beading of intrahepatic ducts (11/12, 92%), bile duct wall thickening with enhancement (7/12, 58%), and peribiliary diffusion high signal (10/12, 83%). Liver biopsy in 4 patients showed acute and/or chronic large duct obstruction without clear bile duct loss. Progressive biliary tract damage has been demonstrated radiographically. Five patients were referred for consideration of liver transplantation after experiencing persistent jaundice, hepatic insufficiency, and/or recurrent bacterial cholangitis. One patient underwent successful living donor liver transplantation., Discussion: Cholangiopathy is a late complication of severe COVID-19 with the potential for progressive biliary injury and liver failure. Further studies are required to understand pathogenesis, natural history, and therapeutic interventions., (Copyright © 2021 by The American College of Gastroenterology.)

Background & Aims: IL-17A-producing T cells are present in autoimmune cholestatic liver diseases; however, little is known about the contribution of IL-17 to periductal immune responses. Herein, we investigated the role of IL-17 produced by antigen-specific CD8 + T cells in a mouse model of cholangitis and in vitro in human cholangiocyte organoids., Methods: K14-OVAp mice express a major histocompatibility complex I-restricted ovalbumin (OVA) peptide sequence (SIINFEKL) on cholangiocytes. Cholangitis was induced by the adoptive transfer of transgenic OVA-specific ovalbumin transgene (OT)-1 CD8 + T cells that either had OT-1 wt or lacked IL-17A/F (OT-1 IL17ko ). The response of mouse and human cholangiocytes/organoids to IL-17A was assessed in vitro., Results: Transfer of OVA-specific OT-1 IL17ko cells significantly aggravated periductal inflammation in K14-OVAp recipient mice compared with transfer of OT-1 wt T cells. OT-1 IL17ko T cells were highly activated in the liver and displayed increased cytotoxicity and proliferation. IL-17A/F produced by transferred OT-1 wt CD8 + T cells induced upregulation of the inhibitory molecule programmed cell death ligand 1 (PD-L1) on cholangiocytes, restricting cholangitis by limiting cytotoxicity and proliferation of transferred cells. In contrast, OT-1 IL17ko T cells failed to induce PD-L1 on cholangiocytes, resulting in uncontrolled expansion of cytotoxic CD8 + T cells and aggravated cholangitis. Blockade of PD-L1 after transfer of OT-1 wt T cells with anti-PD-L1 antibody also resulted in aggravated cholangitis. Using human cholangiocyte organoids, we were able to confirm that IL-17A induces PD-L1 expression in cholangiocytes., Conclusions: We demonstrate that by upregulating PD-L1 on cholangiocytes, IL-17 has an important role in restricting cholangitis and protecting against CD8 + T cell-mediated inflammatory bile duct injury. Caution should be exercised when targeting IL-17 for the treatment of cholangitis., Lay Summary: IL-17 is assumed to be a driver of inflammation in several autoimmune diseases, such as psoriasis. IL-17 is also present in inflammatory diseases of the bile duct, but its role in these conditions is not clear, as the effects of IL-17 depend on the context of its expression. Herein, we investigated the role of IL-17 in an experimental autoimmune cholangitis mouse model, and we identified an important protective effect of IL-17 on cholangiocytes, enabling them to downregulate bile duct inflammation via checkpoint inhibitor PD-L1., Competing Interests: Conflict of interest The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2020 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Atezolizumab is an immune checkpoint inhibitor that is a key drug in non-small-cell lung cancer treatment. However, it can cause immune-related adverse events, including liver injury. Several patterns of liver injury associated with immune checkpoint inhibitor therapy have been reported; however, not much is known about sclerosing cholangitis. We present here a case of lung adenocarcinoma with atezolizumab-induced secondary sclerosing cholangitis diagnosed using needle biopsy of the liver. A 77-year-old woman with lung adenocarcinoma, cT3N2M0, stage IIIA, was treated with concurrent chemoradiotherapy involving carboplatin and paclitaxel, which markedly reduced the tumor diameter. However, 5 months later, the lesion regrew, and she underwent 39 cycles of pemetrexed monotherapy. As pulmonary metastasis progressed, she was treated with atezolizumab. After 13 cycles of atezolizumab therapy, she complained of nausea. Laboratory tests showed elevated levels of the biliary tract and hepatic enzymes. Nevertheless, abdominal computed tomography and ultrasonography revealed no underlying related cause. Ultrasound-guided needle biopsy of the liver was performed, and histopathological analysis of biopsy samples showed features of sclerosing cholangitis. Further examinations were performed, and a diagnosis of atezolizumab-induced secondary sclerosing cholangitis without strictures and dilatations of the large bile ducts was established. Prednisolone was administered orally, after which the biliary tract and hepatic enzyme levels improved immediately. In patients presenting with a hepatic injury during immune checkpoint inhibitor therapy, clinicians should be aware of the possibility of immune checkpoint inhibitor-induced sclerosing cholangitis, even if the large bile ducts have no strictures and dilatations., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Cholangiocarcinoma (CCA) is a common hepatobiliary cancer in East and Southeast Asia. The data of microbiota contribution in CCA are still unclear. Current available reports have demonstrated that an Opisthorchis viverrini (OV) infection leads to dysbiosis in the bile duct. An increase in the commensal bacteria Helicobacter spp. in OV-infected CCA patients is associated with bile duct inflammation, severity of bile duct fibrosis, and cholangiocyte proliferation. In addition, secondary bile acids, major microbial metabolites, can mediate cholangiocyte inflammation and proliferation in the liver. A range of samples from CCA patients (stool, bile, and tumor) showed different degrees of dysbiosis. The evidence from these samples suggests that OV infection is associated with alterations in microbiota and could potentially have a role in CCA. In this comprehensive review, reports from in vitro, in vivo, and clinical studies that demonstrate possible links between OV infection, microbiota, and CCA pathogenesis are summarized and discussed. Understanding these associations may pave ways for novel potential adjunct intervention in gut microbiota in CCA patients., (Copyright © 2020 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The American College of Gastroenterology.)

Background: Biliary atresia (BA) is an inflammatory pediatric cholangiopathy with only surgical means for treatment. Many contributors to bile acid synthesis and transport have previously been reported to be downregulated in patients with BA; yet, the driving factors of the abnormal bile acid synthesis and transport in regard to BA have not been previously studied., Materials and Methods: Wild type or Ig-α -/- mice were injected with salt solution (control) or rotavirus on day of life 0, and analyses were performed on day of life 14. The mRNA levels of bile acid transporters/nuclear receptors and liver microRNAs (miRNAs) were compared between groups. A mouse hepatocyte cell line was used to examine the effects of innate cytokines on miRNA levels and bile acid transporter/nuclear receptor expression and miRNAs on bile acid transporter/nuclear receptor expression., Results: BA mice had significantly increased mRNA expression of innate cytokines and miRNAs known to bind bile acid transporters/nuclear receptors (miRNAs -22-5p, -34a-5p, and -222-3p) and decreased mRNA expression of bile acid transporters and nuclear receptors. In vitro, TNF-α and IL-1β decreased BSEP and CYP7A1 while increasing miRNA-34a-5p and miRNA 222-3p. LXR, SHP, CYP7A1, NTCP, and MRP2 were decreased by miRNA-34a-5p, whereas miRNA-222-3p decreased NTCP and MRP4. TNF-α and IL-1β increased expression of miRNAs 34a-5p and 222-3p and these miRNAs then decrease expression of multiple bile acid transporters and nuclear receptors., Conclusions: Loss of bile acid transporters increases hepatotoxicity via bile acid retention. Therapeutic agents that increase bile acid transport or nuclear receptor functioning should be investigated in BA., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background & Aims: Senescent biliary epithelial cells (BECs) may be involved in the pathophysiology of primary biliary cholangitis (PBC) by secreting senescence-associated secretory phenotypes. We examined an association of the extent of cellular senescence in BECs with clinicopathological features including response to ursodeoxycholic acid (UDCA) and a possibility of senolytic therapy in PBC., Methods: The expression of senescent markers (p21 WAF1/Cip1 , p16 INK4a ) and B-cell lymphoma-extra large (Bcl-xL), a key regulator of senescent cell anti-apoptotic pathway, was immunohistochemically examined in livers from patients with PBC (n = 145) and 103 control livers. Senolytic effect of Bcl-xL inhibitors (A-1331852 and Navitoclax) was examined in senescent murine BECs., Results: Senescent BECs were increased in small bile ducts in PBC, compared with control livers (p < 0.01). Senescent BECs were increased in ductular reactions in PBC, stage 3-4, compared with PBC, stage 1-2 and control livers (p < 0.01). The extent of senescent BECs in bile ductules was significantly correlated with stage and hepatitis activity (p < 0.01) and the expression of p16 INK4a in bile ductules was significantly correlated to inadequate response to UDCA in PBC (p < 0.01). Double immunofluorescence revealed an increased expression of Bcl-xL in p16 INK4a -positive senescent BECs in PBC. Bcl-xL inhibitors selectively induced apoptosis in senescent murine BECs (p < 0.01)., Conclusion: The extent of senescent BECs in small bile ducts and bile ductules was closely related to stage and activity of PBC and the increased expression of p16 INK4a in bile ductules was correlated with inadequate response to UDCA., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Primary biliary cholangitis (PBC) is an autoimmune disease characterized by chronic destruction of the bile ducts. A major unanswered question regarding the pathogenesis of PBC is the precise mechanisms of small bile duct injury. Emperipolesis is one of cell-in-cell structures that is a potential histological hallmark associated with chronic hepatitis B. This study aimed to clarify the pathogenesis and characteristics of emperipolesis in PBC liver injury. Sixty-six PBC patients, diagnosed by liver biopsy combined with laboratory test, were divided into early-stage PBC (stages I and II, n = 39) and late-stage PBC (stages III and IV, n = 27). Emperipolesis was measured in liver sections stained with haematoxylin-eosin. The expressions of CK19, CD3, CD4, CD8, CD20, Ki67 and apoptosis of BECs were evaluated by immunohistochemistry or immunofluorescence double labelling. Emperipolesis was observed in 62.1% of patients with PBC, and BECs were predominantly host cells. The number of infiltrating CD3 + and CD8 + T cells correlated with the advancement of emperipolesis (R 2  = 0.318, P < .001; R 2  = 0.060, P < .05). The cell numbers of TUNEL-positive BECs and double staining for CK19 and Ki67 showed a significant positive correlation with emperipolesis degree (R 2  = 0.236, P < .001; R 2  = 0.267, P < .001). We conclude that emperipolesis mediated by CD8 + T cells appears to be relevant to apoptosis of BEC and thus may aggravate the further injury of interlobular bile ducts., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Background: IgG4-related diseases are a rare but an important entity. Due to the variable clinical presentation, this multiorgan disease was attributed to single-organ systems for many years. Also, it often remains a challenge to differentiate between IgG4-related diseases and malignancies. The pathogenesis seems to be a mixture of Th1- and Th2- immune responses, whereas the role of the non-pathogenic IgG4 antibodies is still unclear. Histopathological characteristics are a lymphoplasmacellular infiltrate with IgG4+ plasma cells, a storiform fibrosis and an obliterative phlebitis. This can lead to the functional destruction of every organ affected. In most cases, glucocorticoid treatment leads to remission and is used as maintenance therapy as well. Immune modulatory therapies are employed in case of steroid resistance. However, a majority of patients achieve remission without any therapy., Summary: In this study, we review the current state-of-the-art regarding pathophysiology, diagnostics, organ manifestation and therapeutic approaches. Key Messages: While the diagnosis of IgG4-related diseases is still challenging, there have been significant improvements in diagnostic as well as in therapeutic approaches. This is partially due to a better understanding of the pathophysiology of the disease but also due to improved imaging modalities and novel, more targeted therapies., (© 2018 S. Karger AG, Basel.)

Juvenile sclerosing cholangitis is a rare chronic hepatobiliary disorder characterized by inflammation of the intra- and/or extrahepatic bile ducts, bile duct dilatation, narrowing and obliteration, and, histologically, by inflammatory bile duct damage leading to periductular fibrosis. The diagnosis is based on endoscopic retrograde cholangiopancreatography or magnetic resonance cholangiopancreatography. In children, it may be associated to a variety of systemic and hepatic conditions: thus, the term "primary" sclerosing cholangitis should be reserved for the rare cases without a known cause. Small duct disease is diagnosed in the presence of histological features diagnostic of sclerosing cholangitis and normal cholangiography. Autoimmune sclerosing cholangitis (ASC) is a form of sclerosing cholangitis with strong autoimmune features overlapping with those of autoimmune hepatitis (AIH). It is a well-recognized nosological entity in paediatrics, where it accounts for the majority of sclerosing cholangitis cases. It is as prevalent as AIH in children, is equally frequent in males and females, half of the patients have concomitant inflammatory bowel disease, virtually all patients have raised immunoglobulin G levels and positive anti-nuclear and/or anti-smooth muscle antibodies. Half of the ASC patients respond well to standard immunosuppressive treatment for AIH with the addition of ursodeoxycholic acid, but the transplant rate is higher than in AIH, and post-transplant recurrence is frequent. A number of open questions remain: are ASC and AIH distinct entities or different manifestations of the same condition? What is the role of histology? Is small duct disease a specific entity? What is the relationship between ASC and adult primary sclerosing cholangitis? What is the role of inflammatory bowel disease? In addition, validated diagnostic criteria for ASC are needed., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Gut microbiota and bacterial translocation have been implicated as significant contributors to mucosal immune responses and tolerance; alteration of microbial molecules, termed pathogen-associated molecular patterns (PAMP) and bacterial translocation are associated with immune pathology. However, the mechanisms by which dysregulated gut microbiota promotes autoimmunity is unclear. We have taken advantage of a well-characterized murine model of primary biliary cholangitis, dnTGFβRII mice, and an additional unique construct, toll-like receptor 2 (TLR2)-deficient dnTGFβRII mice coined dnTGFβRIITLR2 -/- mice to investigate the influences of gut microbiota on autoimmune cholangitis. Firstly, we report that dnTGFβRII mice manifest altered composition of gut microbiota and that alteration of this gut microbiota by administration of antibiotics significantly alleviates T-cell-mediated infiltration and bile duct damage. Second, toll-like receptor 2 (TLR2)-deficient dnTGFβRII mice demonstrate significant exacerbation of autoimmune cholangitis when their epithelial barrier integrity was disrupted. Further, TLR2-deficiency mediates downregulated expression of tight junction-associated protein ZO-1 leading to increased gut permeability and bacterial translocation from gut to liver; use of antibiotics reduces microbiota translocation to liver and also decreases biliary pathology. In conclusion, our data demonstrates the important role of gut microbiota and bacterial translocation in the pathogenesis of murine autoimmune cholangitis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Primary sclerosing cholangitis (PSC) is a severe chronic liver disease of the small and large bile ducts. The pathogenesis is unknown but a strong immune cell component has been suggested. Mucosal-associated invariant T (MAIT) cells are abundant in human liver and localize around bile ducts. Yet, the role of MAIT cells in PSC remains unclear. Here, we performed a detailed characterization of MAIT cells in circulation and assessed their presence in bile ducts of PSC patients as well as non-PSC controls. We observed a dramatic reduction in MAIT cell levels in PSC patients. High-dimensional phenotypical analysis using stochastic neighbor embedding revealed the MAIT cells to be activated, a phenotype shared by the investigated disease control groups. In line with the noted phenotypic alterations, MAIT cell function was reduced in response to Escherichia coli and to cytokine stimulation in PSC patients as compared to healthy controls. Using a novel sampling approach of human bile ducts, we found MAIT cells to be specifically enriched within bile ducts. Finally, distinct from the dramatic decline observed in circulation, PSC-patients had retained levels of MAIT cells within bile ducts. Altogether, our results provide a detailed insight into how the human MAIT cell compartment is affected in PSC., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: Biliary atresia (BA) is the main indication for pediatric liver transplantation. The aim of this study is to correlate aspects of histological examinations of diagnostic hepatic biopsies for BA with the patients' clinical progression and successful addition to the liver transplant waitlist., Methods: This was a retrospective study of all 108 BA cases treated at the Federal University of São Paulo (1998-2015). Demographic and clinical data were correlated with histological findings. A logistic regression was used for outcome analysis, while the Kaplan-Meier method was applied for survival analysis., Results: There were 108 patients with BA, 68.5% of whom underwent Kasai surgery. Patients added to the transplant waitlist tended to undergo Kasai surgery at a later time (P = .035). Periductal lymphocytic infiltrate was correlated with the addition to the transplant waitlist, with an odds ratio of 3.92 (P = .033). Patients who developed ascites after surgery were more frequently added to the transplant waitlist (P = .05)., Conclusion: Patients added to the transplant waitlist underwent Kasai surgery later than other patients. Periductal lymphocytic infiltrate in the diagnostic hepatic biopsy and ascites after Kasai surgery were associated with an increased likelihood of addition to the transplant waitlist., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Biliary atresia (BA) is a neonatal T cell-mediated, inflammatory, sclerosing cholangiopathy. In the rhesus rotavirus (RRV)-induced neonatal mouse model of BA (murine BA), mice lacking B cells do not develop BA, and the lack of B cells is associated with loss of T-cell and macrophage activation. The aim of this study was to determine the mechanism of B cell-mediated immune activation (antigen presentation versus cytokine production) in murine BA. Normal neonatal B cells in the liver are predominantly at pro-B and pre-B cellular development. However, BA mice exhibit a significant increase in the number and activation status of mature liver B cells. Adoptively transferred B cells into RRV-infected, B cell-deficient mice were able to reinstate T-cell and macrophage infiltration and biliary injury. Nonetheless, neonatal liver B cells were incompetent at antigen presentation to T cells. Moreover, 3-83 immunoglobulin transgenic mice, in which B cells only present an irrelevant antigen, developed BA, indicating a B-cell antigen-independent mechanism. B cells from BA mice produced a variety of innate and adaptive immune cytokines associated with immune activation. In vitro trans-well studies revealed that BA B cells secreted cytokines that activated T cells based on increased expression of T-cell activation marker cluster of differentiation 69. Conclusion: Neonatal liver B cells are highly activated in murine BA and contribute to immune activation through production of numerous cytokines involved in innate and adaptive immunity; this work provides increased knowledge on the capacity of neonatal B cells to contribute to an inflammatory disease through cytokine-mediated mechanisms, and future studies should focus on targeting B cells as a therapeutic intervention in human BA., (© 2018 by the American Association for the Study of Liver Diseases.)

Background: Mesenchymal stem cells (MSCs) play an anti-inflammatory role by secreting certain bioactive molecules to exert their therapeutic effects for disease treatment. However, the underlying mechanism of MSCs in chronic autoimmune liver diseases-primary biliary cholangitis (PBC), for example-remains to be elucidated., Methods: Human umbilical cord-derived MSCs (UC-MSCs) were injected intravenously into 2-octynoic acid coupled to bovine serum albumin (2OA-BSA)-induced autoimmune cholangitis mice. Serum levels of biomarkers and autoantibodies, histologic changes in the liver, diverse CD4 +  T-cell subsets in different tissues, and chemokine activities were analyzed. Moreover, we investigated galectin-9 (Gal-9) expression and its function in UC-MSCs., Results: In this study, UC-MSC transplantation (UC-MSCT) significantly ameliorated liver inflammation, primarily by diminishing T helper 1 (Th1) and Th17 responses as well as modifying liver chemokine activities in experimental autoimmune cholangitis mice. Mechanistically, UC-MSCs significantly repressed the proliferation of CD4 + T cells and suppressed the differentiation of Th1 and Th17 cells, which was likely dependent on Gal-9. Furthermore, the signal transducer and activator of transcription (STAT) and c-Jun N-terminal kinase (JNK) signaling pathways were involved in the production of Gal-9 in UC-MSCs., Conclusions: These results suggest that Gal-9 contributes significantly to UC-MSC-mediated therapeutic effects and improve our understanding of the immunomodulatory mechanisms of MSCs in the treatment of PBC.

Gulf War Illness (GWI) is a chronic multisymptom disorder affecting veterans of the 1990-91 Gulf war. GWI was linked with exposure to chemicals including the nerve gas prophylactic drug pyridostigmine-bromide (PB) and pesticides (DEET, permethrin). Veterans with GWI exhibit prolonged, low-level systemic inflammation, though whether this impacts the liver is unknown. While no evidence exists that GWI-related chemicals are hepatotoxic, the prolonged inflammation may alter the liver's response to insults such as cholestatic injury. We assessed the effects of GWI-related chemicals on macrophage infiltration and its subsequent influence on hepatic cholestasis. Sprague Dawley rats were treated daily with PB, DEET and permethrin followed by 15 minutes of restraint stress for 28 days. Ten weeks afterward, GWI rats or naïve age-matched controls underwent bile duct ligation (BDL) or sham surgeries. Exposure to GWI-related chemicals alone increased IL-6, and CD11b + F4/80 - macrophages in the liver, with no effect on biliary mass or hepatic fibrosis. However, pre-exposure to GWI-related chemicals enhanced biliary hyperplasia and fibrogenesis caused by BDL, compared to naïve rats undergoing the same surgery. These data suggest that GWI patients could be predisposed to developing worse liver pathology due to sustained low-level inflammation of the liver when compared to patients without GWI.

Immune responses are critical for defense against pathogens. However, prolonged viral infection can result in defective T cell immunity, leading to chronic viral infection. We studied immune activation in response to arenavirus infection during cholestasis using bile duct ligation (BDL). We monitored T cell responses, virus load and liver pathology markers after infection with lymphocytic choriomeningitis virus (LCMV). BDL mice failed to induce protective anti-viral immunity against LCMV and consequently exhibited chronic viral infection. BDL mice exhibited reduced anti-viral T cell immunity as well as reduced type 1 interferon production early after LCMV infection. Consistently, the presence of serum from BDL mice reduced the responsiveness of dendritic cell (DC) and T cell cultures when compared to Sham controls. Following fractionation and mass spectrometry analyses of sera, we identified several serum factors to be upregulated following BDL including bilirubin, bile acids, 78 kDa Glucose regulated protein (GRP78) and liver enzymes. Bilirubin and GRP78 were capable of inhibiting DC and T cell activation. In this work, we demonstrate that liver damage mediated by cholestasis results in defective immune induction following arenavirus infection.

Cholestatic patients exhibiting high bile acid serum levels were reported to be more susceptible to bacterial and viral infections. Animal studies in bile duct ligated (BDL) mice suggest that cholestasis leads to an aggravation of hepatic bacterial infections. We have investigated the impact of cholestasis on mouse cytomegalovirus (MCMV)-induced immune responses and viral replication. While MCMV did not aggravate BDL-induced liver damage, BDL markedly reduced MCMV-triggered chemokine expression and immune cell recruitment to the liver. MCMV-infected BDL mice showed diminished trafficking of Ly6C+/F4/80+ myeloid cells and NK1.1+ NK cells to the liver compared to MCMV infected control mice. Moreover, virus-driven expression of CCL7, CCL12, CXCL9 and CXCL10 was clearly impaired in BDL- compared to sham-operated mice. Furthermore, production of the anti-inflammatory cytokine IL-10 was massively augmented in infected BDL mice. In contrast, intra- and extrahepatic virus replication was unaltered in BDL-MCMV mice when compared to sham-MCMV mice. Cholestasis in the BDL model severely impaired pathogen-induced chemokine expression in the liver affecting CCR2- and CXCR3-dependent cell trafficking. Cholestasis resulted in reduced recruitment of inflammatory monocytes and NK cells to the liver., Competing Interests: The authors have declared that no competing interests exist.

Background & Aims: Variants at the ABCB4 or MDR2 locus, which encodes a biliary transport protein, are associated with a spectrum of cholestatic liver diseases. Exacerbation of liver disease has been linked to increased hepatic levels of interleukin (IL) 17, yet the mechanisms of this increase are not understood. We studied mice with disruption of Mdr2 to determine how defects in liver and alteration in the microbiota contribute to production of IL17 by intrahepatic γδ T cells., Methods: We performed studies with Mdr2 -/- and littermate FVB/NJ (control) mice. IL17 was measured in serum samples by an enzyme-linked immunosorbent assay. Mice were injected with neutralizing antibodies against the γδ T-cell receptor (TCR; anti-γδ TCR) or mouse IL17A (anti-IL17A). Livers were collected and bacteria were identified in homogenates by culture procedures; TCRγδ + cells were isolated by flow cytometry. Fecal samples were collected from mice and analyzed by 16S ribosomal DNA sequencing. Cells were stimulated with antibodies or bacteria, and cytokine production was measured. We obtained tissues from 10 patients undergoing liver transplantation for primary sclerosing cholangitis or chronic hepatitis C virus infection. Tissues were analyzed for cytokine production by γδ TCR + cells., Results: Mdr2 -/- mice had collagen deposition around hepatic bile ducts and periportal-bridging fibrosis with influx of inflammatory cells and increased serum levels of IL17 compared with control mice. Administration of anti-IL17A reduced hepatic fibrosis. Livers from Mdr2 -/- mice had increased numbers of IL17A + γδTCR + cells-particularly of IL17A + Vγ6Jγ1 γδ TCR + cells. Fecal samples from Mdr2 -/- mice were enriched in Lactobacillus, and liver tissues were enriched in Lactobacillus gasseri compared with control mice. Mdr2 -/- mice also had increased intestinal permeability. The γδ TCR + cells isolated from Mdr2 -/- livers produced IL17 in response to heat-killed L gasseri. Intraperitoneal injection of control mice with L gasseri led to increased serum levels of IL17 and liver infiltration by inflammatory cells; injection of these mice with anti-γδ TCR reduced serum level of IL17. Intravenous injections of Mdr2 -/- mice with anti-γδ TCR reduced fibrosis; liver levels of IL17, and inflammatory cells; and serum levels of IL17. γδTCR + cells isolated from livers of patients with primary sclerosing cholangitis, but not hepatitis C virus infection, produced IL17., Conclusions: In Mdr2 -/- mice, we found development of liver fibrosis and inflammation to require hepatic activation of γδ TCR + cells and production of IL17 mediated by exposure to L gasseri. This pathway appears to contribute to development of cholestatic liver disease in patients., (Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The most studied physiological function of biliary epithelial cells (cholangiocytes) is to regulate bile flow and composition, in particular the hydration and alkalinity of the primary bile secreted by hepatocytes. After almost three decades of studies it is now become clear that cholangiocytes are also involved in epithelial innate immunity, in inflammation, and in the reparative processes in response to liver damage. An increasing number of evidence highlights the ability of cholangiocyte to undergo changes in phenotype and function in response to liver damage. By participating actively to the immune and inflammatory responses, cholangiocytes represent a first defense line against liver injury from different causes. Indeed, cholangiocytes express a number of receptors able to recognize pathogen- or damage-associated molecular patterns (PAMPs/DAMPs), such as Toll-like receptors (TLR), which modulate their pro-inflammatory behavior. Cholangiocytes can be both the targets and the initiators of the inflammatory process. Derangements of the signals controlling these mechanisms are at the basis of the pathogenesis of different cholangiopathies, both hereditary and acquired, such as cystic fibrosis-related liver disease and sclerosing cholangitis. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Introduction: Endotoxins, in the form of lipopolysaccharides (LPS), are potent inducers of biliary injury. However the mechanism by which injury develops remains unclear. We hypothesized that hepatic macrophages are pivotal in the development of endotoxin-induced biliary injury and that no injury would occur in their absence., Material and Methods: Clodronate liposomes were used to deplete macrophages from the liver. Forty-eight rats were equally divided across six study groups: sham operation (sham), liposome treatment and sham operation (liposomes+sham), 1mg/kg LPS i.p. (LPS), liposome treatment and LPS administration (liposomes+LPS), hepatic ischaemia-reperfusion injury with LPS administration (IRI+LPS) and liposome treatment followed by IRI+LPS (liposomes+IRI+LPS). Following 6h of reperfusion, blood, bile, and liver tissue was collected for further analysis. Small bile duct injury was assessed, serum liver tests were performed and bile composition was evaluated. The permeability of the blood-biliary barrier (BBB) was assessed using intravenously administered horseradish peroxidase (HRP)., Results: The presence of hepatic macrophages was reduced by 90% in LPS and IRI+LPS groups pre-treated with clodronate liposomes (P<0.001). Severe small bile duct injury was not affected by macrophage depletion, and persisted in the liposomes+IRI+LPS group (50% of animals) and liposomes+LPS group (75% of animals). Likewise, BBB impairment persisted following macrophage depletion. LPS-induced elevation of the chemokine Mcp-1 in bile was not affected by macrophage depletion., Conclusions: Depletion of hepatic macrophages did not prevent development of biliary injury following LPS or LPS-enhanced IRI. Cholangiocyte activation rather than macrophage activation may underlie this injury. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The pathophysiology of immunoglobulin G4-related disease (IgG4-RD) and its most common manifestations, IgG4-associated (sclerosing) cholangitis and autoimmune pancreatitis, remains largely unknown, but IgG4 is presumably involved. IgG4 is a promiscuous antibody, which could be directly pathogenic, fulfill a protective role, or could just be a fortuitous marker of an aberrant inflammatory response. IgG4 antibodies possess exclusive structural and functional characteristics suggesting anti-inflammatory and tolerance-inducing effects. By studying the role of IgG4 in other inflammatory conditions, namely hypersensitivity and allergies, autoimmune and immune-mediated diseases, infections and malignancies, new insights can be obtained increasing our understanding of the role of IgG4 antibodies in IgG4-RD. Beekeepers, animal laboratory workers and individuals undergoing allergen immunotherapy possess high serum levels of allergen-specific IgG4, which exhibit immunosuppressive functions, protecting the individual from anaphylactic reactions. In autoimmune/immune-mediated diseases, such as pemphigus vulgaris, pemphigus foliaceus and MuSK-myasthenia gravis, IgG4 autoantibodies are pathogenic. Regarding malignancies such as melanoma and cholangiocarcinoma or helminthic infections, IgG4 antibodies inhibit clearance of tumor cells or the invader, respectively. Translating these findings to IgG4-RD, IgG4 alone can implement pathogenic effects and structural damage, but may also function as a protective antibody dampening the more harmful effects of IgG1 when directed against the same epitopes. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Primary biliary cholangitis (PBC) is a chronic fibrosing cholangiopathy characterized by an autoimmune stereotype and defective biliary bicarbonate secretion due to down-regulation of anion exchanger 2 (AE2). Despite the autoimmune features, immunosuppressants are ineffective while two bile acid-based therapies (ursodeoxycholic acid and obeticholic acid) have been shown to improve biochemical and histological features of cholestasis and long-term prognosis. However, the etiology and pathogenesis of PBC is largely unknown. Recently, it has been shown that microRNA-506 (miR-506) on chromosome X is up-regulated in PBC cholangiocytes and suppresses AE2 expression, which sensitizes cholangiocytes to bile salt-induced apoptosis by activating soluble adenylyl cyclase (sAC), an evolutionarily conserved bicarbonate sensor. In this review, we discuss the experimental evidence for the emerging role of the miR-506-AE2-sAC axis in PBC pathogenesis. We further hypothesize that the initial disease trigger induces an X-linked epigenetic change, leading to a female-biased activation of the miR-506-AE2-sAC axis. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni and Peter Jansen., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Primary sclerosing cholangitis (PSC) is an idiopathic cholangiopathy strongly associated with inflammatory bowel disease (IBD) and characterized by cholestasis, chronic immune infiltration and progressive fibrosis of the intrahepatic and extrahepatic bile ducts. PSC confers a high risk of cholangiocarcinoma (CCA) with PSC-CCA representing the leading cause of PSC-associated mortality. PSC-CCA is derived from cholangiocytes and associated progenitor cells - a heterogeneous group of dynamic epithelial cells lining the biliary tree that modulate the composition and volume of bile production by the liver. Infection, inflammation and cholestasis can trigger cholangiocyte activation leading to an increased expression of adhesion and antigen-presenting molecules as well as the release of various inflammatory and fibrogenic mediators. As a result, activated cholangiocytes engage in a myriad of cellular processes, including hepatocellular proliferation, apoptosis, angiogenesis and fibrosis. Cholangiocytes can also regulate the recruitment of immune cells, mesenchymal cells, and endothelial cells that participate in tissue repair and destruction in settings of persistent inflammation. In PSC, the role of cholangiocytes and the mechanisms governing their transformation to PSC-CCA are unclear however localization of disease suggests that cholangiocytes are a key target and potential regulator of hepatobiliary immunity, fibrogenesis and tumorigenesis. Herein, we summarize mechanisms of cholangiocyte activation in PSC and highlight new insights into disease pathways that may contribute to the development of PSC-CCA. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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 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 expressing human leukocyte antigen molecules and costimulatory molecules; recruit leukocytes to the target site by expressing cytokines and chemokines. However, breach of tolerance in the biliary tree results in various cholangiopathies, exemplified by primary biliary cholangitis, primary sclerosing cholangitis and biliary atresia. Lessons learned from immune tolerance of the biliary tree will provide the basis for the development of effective therapeutic approaches against autoimmune biliary tract diseases. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Chronic cholestatic liver diseases such as primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) are associated with active hepatic fibrogenesis, and, ultimately, to the development of cirrhosis. However, the precise relationship between cholestasis, in its broad meaning, and liver tissue fibrosis is still poorly defined. Fibrogenesis is currently viewed as a dynamic process that appears strictly related to the extent and duration of parenchymal injury. This relationship is clearly evident in the presence of reiterative hepatocellular necrosis due to viral infection or alcohol abuse. It appears that "pure" intralobular intrahepatic cholestasis secondary to biliary secretory failure of the hepatocyte, in absence of hepatocellular damage, lobular inflammation and bile duct damage and/or proliferation, is not associated with marked and/or progressive liver tissue fibrosis. In contrast, marked and progressive liver tissue fibrosis always follows liver diseases characterized by chronic inflammatory bile duct damage as seen in PBC and PSC or chronic mechanical obstruction of the biliary tree. Overall, the fibrogenic process in these clinical conditions appears to be related to a more complex interaction between immune/inflammatory mechanisms, cytokine networks and the derangement of the homeostasis between epithelial and mesenchymal cells. The elucidation of these mechanisms is indeed crucial for the identification of potential diagnostic and therapeutic targets., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Primary biliary cholangitis (PBC) is an autoimmune liver disease characterized by the destruction of interlobular biliary ductules, which progressively leads to cholestasis, hepatic fibrosis, cirrhosis, and eventually liver failure. Several mouse models have been used to clarify the pathogenesis of PBC and are generally considered reflective of an autoimmune cholangitis. Most models focus on issues of molecular mimicry between the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), the major mitochondrial autoantigen of PBC and xenobiotic cross reactive chemicals. None have focused on the classic models of breaking tolerance, namely immunization with self-tissue. Here, we report a novel mouse model of autoimmune cholangitis via immunization with syngeneic bile duct protein (BDP). Our results demonstrate that syngeneic bile duct antigens efficiently break immune tolerance of recipient mice, capturing several key features of PBC, including liver-specific inflammation focused on portal tract areas, increased number and activation state of CD4 and CD8 T cells in the liver and spleen. Furthermore, the germinal center (GC) responses in the spleen were more enhanced in our mouse model. Finally, these mice were 100% positive for anti-mitochondrial antibodies (AMAs). In conclusion, we developed a novel mouse model of PBC that may help to elucidate the detailed mechanism of this complex disease.

Bile acids (BA) are key molecules in generating bile flow, which is an essential function of the liver. In the last decades there have been great advances in the understanding of the role of a number of specific transport proteins present at the sinusoidal and canalicular membrane domains of hepatocytes and cholangiocytes in generating and maintaining bile flow. Also, a clearer understanding on how BA regulate their own synthesis and the expression and/or function of transporters has been reached. This new knowledge has helped to better delineate the pathophysiology of cholestasis and the adaptive responses of hepatocytes to cholestatic liver injury as well as of the mechanisms of injury of biliary epithelia. In this context, therapeutic approaches including new hydrophilic BA such as the conjugation-resistant nor- ursodeoxycholic acid, nuclear receptors (FXR, PPAR-alpha) agonists, FGF19 analogues, inhibitors of the apical sodium-dependent bile acid transporter [ASBT] and modulators of the inflammatory cascade triggered by BAs are being studied as novel treatments of cholestasis. In the present review we summarize recent experimental and clinical data on the role of BAs in cholestasis and its treatment.

Purpose: A proposed etiopathogenesis of biliary atresia (BA) involves T-cell-mediated inflammatory bile duct damage and progressive hepatic fibrosis. Pediatric surgeons often observe swelling of the hepatic hilar lymph nodes during the Kasai procedure. Given the importance of regulatory mechanisms in immune responses, the present study was designed to analyze the quantitative changes of regulatory T cells (T reg cells) in the hepatic hilar lymph nodes (hepatic hilar LNs) and peripheral blood (PB) in BA., Methods: The hepatic hilar LNs and PB obtained during the Kasai procedure were analyzed by flow cytometry. The ratios of total and active Tregs to the total CD4 + cells in the PB and the hepatic hilar LNs were compared., Results: In patients with BA, the ratios of both the total and active T reg cells in the hepatic hilar LNs were higher than those in the PB (total T reg cells: PB vs. LN; P < 0.001; active T reg cells: PB vs. LN; P = 0.001). In BA patients, the increase in the ratio of active T reg cells to the CD4 + cells in the LNs in comparison to the PB was greater than that in control patients. The ratio observed in the BA patients was almost double the ratio observed in the control patients. The median LN/PB ratio in the BA patients was 3.1, while that in controls was 1.6 (P = 0.03)., Conclusion: The present study showed that the ratios of both total T reg cells and active T reg cells were higher in the hepatic hilar lymph nodes of BA patients. This finding could shed light on the pathogenesis of BA.

Background: IgG4-related sclerosing cholangitis (IgG4-SC) is the biliary manifestation of IgG4-related disease (IgG4-RD) but the presence of IgG4-SC in the porta hepatis is difficult to differentiate from hilar cholangiocarcinoma (HCCA). IgG4-related autoimmune hepatitis (IgG4-related AIH) is extremely rare and it is not fully clear whether IgG4-related AIH is a hepatic manifestation of IgG4-RD or a subtype of AIH., Case Presentation: We present a rare case of a 52-year-old male who was admitted with obstructive jaundice and itchy skin. He primarily presented a severe bile duct stricture in the porta hepatis and an elevated serum level of carbohydrate antigen 19-9 (CA19-9) mimicking HCCA. The patient underwent a surgical resection of the left hepatic lobular and cholecyst as well as common bile duct with a right hepatico-jejunostomy. He was finally diagnosed as IgG4-SC accompanied with IgG4-related AIH by immunohistochemistry, but he lacked conventional autoantibodies. The patient responded well to steroid therapy and remains healthy with no signs of recurrence at six-month follow-up., Conclusion: This is the first case report that hepatic portal IgG4-SC overlapping with IgG4-related AIH without the presence of conventional autoantibodies. Additionally, we suggest that IgG4-RD should be always considered in case of a bile duct stricture in the porta hepatis to avoid unnecessary surgical operation., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Primary biliary cholangitis (PBC), formerly known as primary biliary cirrhosis, is an organ-specific autoimmune disease that predominantly affects middle-aged women and is characterized by the chronic progressive destruction of small intrahepatic bile ducts with portal inflammation and, ultimately, fibrosis. The serological hallmark of PBC is the presence of anti-mitochondrial autoantibodies (AMA). Several mechanisms have been proposed for immune-mediated bile duct damage in PBC, including the roles of T cells, B cells, other cell phenotypes, and AMA. A sign of fragility of biliary epithelial cells caused by apoptosis, senescence, and autophagy has also been noted. Several complex steps and mechanisms appear to be involved in the induction and progression of cholangitis and biliary degeneration in patients with PBC. J. Med. Invest. 64: 7-13, February, 2017.

Primary biliary cholangitis (PBC; previously "primary biliary cirrhosis") is a cholestatic, putatively autoimmune-mediated liver disease with a clear female preponderance affecting the intrahepatic small and medium-size bile ducts and resulting in bile duct destruction, ductopenia and portal fibrosis that progresses slowly to biliary cirrhosis. Despite suboptimal response in one third of patients treated with ursodeoxycholic acid (UDCA), this remains the only FDA-approved agent for this disease. In this review, we cover recent advances in research that have yielded numerous agents currently at different stages of the drug pipeline, some of which are expected to be approved in the near future. We also discuss accumulating evidence supporting the use of older agents (fibrates and glucocorticoids) as an adjunctive therapy to UDCA in non-responsive patients. We suggest that with the imminent expansion of the therapeutic armamentarium for PBC, a more comprehensive approach - ideally taking into account not only biochemical markers of disease stage - is needed to better select patients in whom these strategies might be most useful. Studies are also needed to compare the relative efficacy of different proposed second-line treatments not only against UDCA monotherapy., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Apart from their pivotal role in dietary lipid absorption and cholesterol homeostasis, bile acids (BAs) are increasingly recognised as important signalling molecules in the regulation of systemic endocrine functions. As such BAs are natural ligands for several nuclear hormone receptors and G-protein-coupled receptors. Through activating various signalling pathways, BAs not only regulate their own synthesis, enterohepatic recirculation and metabolism, but also immune homeostasis. This makes BAs attractive therapeutic agents for managing metabolic and inflammatory liver disorders. Recent experimental and clinical evidence indicates that BAs exert beneficial effects in cholestatic and metabolically driven inflammatory diseases. This review elucidates how different BAs function as pathogenetic factors and potential therapeutic agents for inflammation-driven liver diseases, focusing on their role in regulation of inflammation and immunity.

There is a significant unmet need in the treatment of primary biliary cirrhosis (PBC) despite significant data on the effector pathways that lead to biliary duct damage. We focused attention on a murine model of PBC, the dominant negative transforming growth factor β receptor II (Tg) mice. To further define the pathways that lead to biliary pathology in these mice, we developed Tg mice deleted of CD4 cells (CD4(-/-)Tg). Interestingly, these mice developed more severe cholangitis than control Tg mice. These mice, which lack CD4 cells, manifested increased levels of IFN-γ produced by effector CD8 cells. It appears that increased cholangitis is due to the absence of CD4 Treg cells. Based on these data, we parabiosed CD4(-/-)Tg mice with established disease at 8-9 weeks of age with C57BL/6 control mice. Such parabiotic "twins" had a significant reduction in autoimmune cholangitis, even though they had established pathology at the time of surgery. We prepared mixed bone marrow chimera mice constructed from CD4(-/-)Tg and CD8(-/-) mice and not only was cholangitis improved, but a decrease in terminally differentiated CD8(+) T effector cells in the presence of wild type CD4 cells was noted. In conclusion, "correcting" the CD4 T cell subset, even in the presence of pathogenic CD8 T cells, is effective in treating autoimmune cholangitis., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Efficacy of the ozonotherapy application as an important component of complex treatment in purulent cholangitis (PCH) was studied. In choledocholithiasis (without infectioning of bile) ozonotherapy may be prescribed as additional component at complex treatment. In PCH ozonotherapy application have promoted the improvement of laboratory indices in 6.6 times, comparing with such, occurring after basic therapy.

Unlabelled: Cholangiocytes express antigen-presenting molecules, but it has been unclear whether they can present antigens. Natural killer T (NKT) cells respond to lipid antigens presented by the major histocompatibility complex class I-like molecule CD1d and are abundant in the liver. We investigated whether cholangiocytes express CD1d and present lipid antigens to NKT cells and how CD1d expression varies in healthy and diseased bile ducts. Murine and human cholangiocyte cell lines as well as human primary cholangiocytes expressed CD1d as determined by flow cytometry and western blotting. Murine cholangiocyte cell lines were able to present both exogenous and endogenous lipid antigens to invariant and noninvariant NKT cell hybridomas and primary NKT cells in a CD1d-dependent manner. A human cholangiocyte cell line, cholangiocarcinoma cell lines, and human primary cholangiocytes also presented exogenous CD1d-restricted antigens to invariant NKT cell clones. CD1d expression was down-regulated in the biliary epithelium of patients with late primary sclerosing cholangitis, primary biliary cirrhosis, and alcoholic cirrhosis compared to healthy controls., Conclusions: Cholangiocytes express CD1d and present antigens to NKT cells and CD1d expression is down-regulated in diseased biliary epithelium, findings which show that the biliary epithelium can activate an important lymphocyte subset of the liver. This is a potentially important immune pathway in the biliary system, which may be capable of regulating inflammation in the context of biliary disease., (© 2015 by the American Association for the Study of Liver Diseases.)

Several cell types are considered to be effector cells in bile duct injury in rhesus rotavirus (RRV)-induced experimental biliary atresia (BA). Here, we identified an increased T helper 17 (Th17) cell population in a BA mode. By depleting the Th17 cells, the BA symptoms (onset of jaundice, acholic stools and retarded growth) were attenuated and the survival rate was improved. Furthermore, we found that in mice with BA, the percentage of CD4+CD25highFoxp3+ T regulatory (Treg) cells decreased along with the increased percentage of Th17 cells. However, the absolute numbers of Treg and Th17 cells were both increased in liver of RRV-injected mice compared to saline-injected mice. The proportion of Th17 cells at 7 days post-infection was decreased if Treg cells isolated from normal adult mice, but not Treg cells from the livers of mice with BA, were intraperitoneally transferred on day 5 of life. In vitro experiments also showed that Treg cells from mice with BA had a diminished suppressive effect on Th17 cell generation. To determine the mechanisms, we investigated the production of cytokines in the liver. The level of IL-6, which has been shown to be abundantly secreted by activated dendritic cells (DCs), was remarkably elevated. Importantly, in a Treg/Th17 cell suppression assay, IL-6 was demonstrated to paralyze the Treg cells' suppressive effect on Th17 cells and eventually the unrestrained increase of Th17 cells contributed to bile duct injury. In conclusion, the DC-regulated Treg-Th17 axis, probably in conjunction with other effector T cells, aggravates progressive inflammatory injury at the time of ductal obstruction.

Primary biliary cirrhosis (PBC) is a chronic progressive autoimmune cholestatic liver disease characterized by highly specific antimitochondrial antibodies (AMAs) and the specific immune-mediated injury of small intrahepatic bile ducts. Unique apoptotic feature of biliary epithelial cells (BECs) may contribute to apotope presentation to the immune system, causing unique tissue damage in PBC. Perpetuation of inflammation may result in senescence of BECs, contributing to irreversible loss of bile duct. In addition to the classic liver manifestations, focal inflammation and tissue damage are also seen in salivary glands and urinary tract in a significant proportion of PBC patients. These findings provide potent support to the idea that molecular mimicry may be involved in the breakdown of autoimmune tolerance and mucosal immunity may lead to a systematic epithelitis in PBC patients. Thus, PBC is considered a generalized epithelitis in clinical practice.

Biliary atresia (BA) is an infantile inflammatory cholangiopathy of unknown etiology although epidemiologic studies and animal models utilizing rotavirus (RV) have suggested a role for viral infection. Proinflammatory and profibrotic cytokines have been detected in infants with BA. The purpose of our study was to investigate the susceptibility of human cholangiocytes (H69 cells) to infection with RRV and to determine if this infection resulted in cytokine secretion. Infection of H69 cells by RRV was noncytolytic and resulted in a time-dependent increase in the release of both infectious virions and cytokines IL-6 and IL-8 into the supernate. The greatest difference in cytokine supernatant levels between infected and mock-infected cells was noted at 24 hours postinfection (h p.i.) for IL-8, 556 ± 111 versus 77 ± 68 pg/mL (p < 0.0001), and at 48 h p.i. for IL-6, 459 ± 64 versus 67 ± 2 pg/mL (p < 0.0001). Production of both cytokines following RRV infection was significantly reduced by pretreating the H69 cells with inhibitors of mitogen-activated protein kinase (MAPK). Conclusion. RRV can infect human cholangiocytes resulting in the production of proinflammatory and profibrotic cytokines via the MAPK pathway. RRV-infected H69 cells could be a useful model system for investigating the viral hypothesis of BA.

Aims: Serum antimitochondrial antibodies are characteristic in most patients with primary biliary cirrhosis (PBC); however, the significance of antimitochondrial antibodies in the pathogenesis of PBC remains unclear. We examined the extent and types of mitochondrial protein-expressing inflammatory cells and its association with deregulated autophagy of mitochondria in biliary epithelial lesions in PBC., Methods: We examined the expression of pyruvate dehydrogenase complex-E2 component and a mitochondrial protein cytochrome c oxidase, subunit I in inflammatory cells in livers taken from patients with PBC (n=35) and control livers (n=64) including primary sclerosing cholangitis. Mitochondrial protein-expressing inflammatory cells were characterised by double immunofluorescence with surface markers., Results: Infiltration of mitochondrial protein-expressing inflammatory cells was mainly observed in portal tracts and in the biliary epithelial layer and around the damaged small bile ducts in PBC. The extent of infiltration in portal tracts was significantly higher in PBC and early stage of chronic viral hepatitis than normal livers. The extent of infiltration around bile ducts and in biliary epithelial layer was significantly higher in early stage of PBC than control livers. Mitochondrial protein-expressing inflammatory cells included (1) CD68 and/or myeloperoxidase -positive monocytes/macrophages and (2) CD79a, CD38, CD138, IgM-positive and/or IgG-positive plasma cells. Colocalised expression of pyruvate dehydrogenase complex-E2 component and autophagy marker light chain 3β was observed in the inflammatory cells., Conclusions: Mitochondrial protein-expressing inflammatory cells infiltrating around the damaged bile ducts and in biliary epithelial layers may be closely associated with the pathogenesis of bile duct lesion in PBC., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Background & Aims: Biliary atresia represents obstructive cholangiopathy in infants progressing rapidly to cirrhosis and end-stage liver disease. Activated NK cells expressing Nkg2d have been linked to bile duct injury and obstruction by establishing contact with cholangiocytes. To define the mechanisms used by cytotoxic cells, we investigated the role of perforin and granzymes in a neonatal mouse model of rotavirus (RRV)-induced biliary atresia., Methods: We used complementary cell lysis assays, flow cytometric analyses, quantitative PCRs and in vivo systems to determine the mechanisms of bile duct epithelial injury and the control of the tissue phenotype in experimental biliary atresia., Results: RRV-infected hepatic NK and CD8 T cells increased the expression of perforin and injured cholangiocytes in short-term culture in a perforin-dependent fashion. However, the loss of perforin in vivo delayed but did not prevent the obstruction of bile ducts. Based on the increased expression of granzymes by perforin-deficient cytotoxic cells in long-term cytolytic assays, we found that the inhibition of granzymes by nafamostat mesilate (FUT-175) blocked cholangiocyte lysis. Administration of FUT-175 to perforin-deficient mice after RRV infection decreased the development of jaundice, minimized epithelial injury, and improved long-term survival. However, the inhibition of granzymes alone in wild-type mice was not sufficient to prevent the atresia phenotype in newborn mice. In infants with biliary atresia, hepatic Granzymes A and B mRNA, but not Perforin, increased at the time of portoenterostomy., Conclusions: Perforin and granzymes have complementary roles mediating epithelial injury by NK and CD8 T cells. The prevention of experimental biliary atresia can only be achieved by inhibiting both granules., (Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background and Aims: Senescent cells can alter local tissue environments by secretion of various senescence-associated secretory phenotypes (SASP), such as cytokines and chemokines. Given senescent biliary epithelial cells (BECs) in damaged small bile ducts in primary biliary cirrhosis (PBC) show increased expression of chemokines CCL2 and CX3CL1 as SASP, we further examined an involvement of CCL2/CCR2 and CX3CL1/CX3CR1 systems in the pathogenesis of PBC., Methods: We examined immunohistochemically the expression of CCR2, CX3CR1, CCL2 and CX3CL1 in livers taken from the patients with PBC (n = 45) and control livers (n = 78), such as chronic viral hepatitis (CVH; n = 39). CCR2 or CX3CR1-expressing cells were characterized by double immunofluorescence with CD3, CD4, CD8, CD56 or CD68., Results: CCR2 is expressed in round cells, epithelioid cells and dendritic cells and most CCR2-positive cells were CD68-positive. Infiltration of CCR2-positive cells in the intraepithelial layer or around small bile ducts was significantly more extensive in PBC than CVH and normal liver (p < 0.05) and was significantly correlated with the expression of CCL2 in BECs (p < 0.01). Most CX3CR1-expressing inflammatory cells were CD3-positive T cells (CD8 > CD4). Infiltration of CX3CR1-positive cells in the intraepithelial layer and around small bile ducts was significantly more extensive in PBC than control livers (p < 0.05) and was significantly correlated with the expression of CX3CL1 in BECs (p < 0.05)., Conclusion: CCL2 and CX3CL1 produced by senescent BECs may promote infiltration of corresponding CCR2 and CX3CR1-expressing cells and further aggravate inflammation in bile duct lesion in PBC.

Aim: To investigate the pathogenesis of biliary casts after liver transplantation relative to their morphology and biochemical markers., Methods: The microstructure of biliary casts was assessed using scanning electron microscopy and Hematoxylin and eosin staining assessed their histology. The expression levels of CD3, CD5, CD34, CD68 and CD79a in these biliary casts were evaluated immunohistochemically., Results: Biliary casts differed widely in their microstructure, with some containing blood vessels positive for CD34 and collagen fibers with positive Masson staining. Large numbers of neutrophils and other inflammatory cells were present, but only on the edge of the biliary casts; although the boundaries were clear without crossover. None of the biliary casts contained T-lymphocytes, B-lymphocytes, macrophages and other inflammatory cells., Conclusion: The microcostructure of biliary casts differed. Bacteria and acute rejection are not clearly related to their formation.

Background & Aims: Biliary atresia (BA) is a pediatric inflammatory disease of the biliary system which leads to cirrhosis and the need for liver transplantation. One theory regarding etiology is that bile duct injury is due to virus-induced autoreactive T cell-mediated inflammation. Regulatory T cell (Treg) abnormalities in BA could result in unchecked bystander inflammation and autoimmunity targeting bile ducts. The aim of this study was to determine if Tregs are dysfunctional in the rotavirus-induced mouse model of BA (murine BA)., Methods: Murine BA resulted from infection of BALB/c neonates with Rhesus rotavirus (RRV)., Results: Liver Tregs from BA mice were decreased in number, activation marker expression, and suppressive function. Adoptive transfer studies revealed that RRV-infected mice that received Tregs had significantly increased survival (84%) compared to controls (12.5%). In addition, ablation of Tregs in older mice, followed by RRV infection, resulted in increased bile duct injury., Conclusions: These studies demonstrate that dysregulation of Tregs is present in murine BA and that diminished Treg function may be implicated in the pathogenesis of human BA., (Published by Elsevier B.V.)

Biliary atresia (BA) is thought to be associated with infections by viruses such as Reoviridae and is characterized histologically by fibrosclerosing cholangitis with proinflammatory cytokine-mediated inflammation. Interleukin (IL)-32 affects the continuous inflammation by increasing the production of proinflammatory cytokines. In this study, the role of IL-32 in the cholangitis of BA was examined. Immunohistochemistry for IL-32 and caspase 1 was performed using 21 samples of extrahepatic bile ducts resected from BA patients. Moreover, using cultured human biliary epithelial cells (BECs), the expression of IL-32 and its induction on stimulation with a Toll-like receptor [(TLR)-3 ligand (poly(I:C)] and proinflammatory cytokines was examined. BECs composing extrahepatic bile ducts showing cholangitis expressed IL-32 in BA, but not in controls. Caspase 1 was expressed constantly on BECs of both BA and control subjects. Furthermore, poly(I:C) and proinflammatory cytokines [(IL-1β, interferon (IFN)-γ and tumour necrosis factor (TNF)-α] induced IL-32 expression strongly in cultured BECs, accompanying the constant expression of TLR-3 and caspase 1. Our results imply that the expression of IL-32 in BECs was found in the damaged bile ducts of BA and induced by biliary innate immunity via TLR-3 and proinflammatory cytokines. These findings suggest that IL-32 is involved initially in the pathogenic mechanisms of cholangitis in BA and also plays an important role in the amplification and continuance of periductal inflammatory reactions. It is therefore tempting to speculate that inhibitors of IL-32 could be useful for attenuating cholangitis in BA., (© 2013 British Society for Immunology.)