IgG4-related cholangitis - a mimicker of fibrosing and malignant cholangiopathies.

IgG4-related cholangitis (IRC) is the major hepatobiliary manifestation of IgG4-related disease (IgG4-RD), a systemic fibroinflammatory disorder. The pathogenesis of IgG4-RD and IRC is currently viewed as multifactorial, as there is evidence of a genetic predisposition while environmental factors, such as blue-collar work, are major risk factors. Various autoantigens have been described in IgG4-RD, including annexin A11 and laminin 511-E8, proteins which may exert a partially protective function in cholangiocytes by enhancing secretion and barrier function, respectively. For the other recently described autoantigens, galectin-3 and prohibitin 1, a distinct role in cholangiocytes appears less apparent. In relation to these autoantigens, oligoclonal expansions of IgG4+ plasmablasts are present in patients with IRC and disappear upon successful treatment. More recently, specific T-cell subtypes including regulatory T cells, follicular T helper 2 cells, peripheral T helper cells and cytotoxic CD8+ and CD4+ SLAMF7+ T cells have been implicated in the pathogenesis of IgG4-RD. The clinical presentation of IRC often mimics other biliary diseases such as primary sclerosing cholangitis or cholangiocarcinoma, which may lead to inappropriate medical and potentially invalidating surgical interventions. As specific biomarkers are lacking, diagnosis is made according to the HISORt criteria comprising histopathology, imaging, serology, other organ manifestations and response to therapy. Treatment of IRC aims to prevent or alleviate organ damage and to improve symptoms and consists of (i) remission induction, (ii) remission maintenance and (iii) long-term management. Glucocorticosteroids are highly effective for remission induction, after which immunomodulators can be introduced for maintenance of remission as glucocorticosteroid-sparing alternatives. Increased insight into the pathogenesis of IRC will lead to improved diagnosis and novel therapeutic strategies in the future.

Role of the IgG4-related cholangitis autoantigen annexin A11 in cholangiocyte protection.

BACKGROUND & AIMS: Annexin A11 was identified as autoantigen in IgG4-related cholangitis (IRC), a B-cell driven disease. Annexin A11 modulates calcium-dependent exocytosis, a crucial mechanism for insertion of proteins into their target membranes. Human cholangiocytes form an apical 'biliary bicarbonate umbrella' regarded as defense against harmful hydrophobic bile acid influx. The bicarbonate secretory machinery comprises the chloride/bicarbonate exchanger AE2 and the chloride channel ANO1. We aimed to investigate the expression and function of annexin A11 in human cholangiocytes and a potential role of IgG1/IgG4-mediated autoreactivity against annexin A11 in the pathogenesis of IRC. METHODS: Expression of annexin A11 in human liver was studied by immunohistochemistry and immunofluorescence. In human control and ANXA11 knockdown H69 cholangiocytes, intracellular pH, AE2 and ANO1 surface expression, and bile acid influx were examined using ratio microspectrofluorometry, cell surface biotinylation, and 22,23-3H-glycochenodeoxycholic acid permeation, respectively. The localization of annexin A11-mEmerald and ANO1-mCherry was investigated by live-cell microscopy in H69 cholangiocytes after incubation with IRC patient serum containing anti-annexin A11 IgG1/IgG4-autoantibodies or disease control serum. RESULTS: Annexin A11 was strongly expressed in human cholangiocytes, but not hepatocytes. Knockdown of ANXA11 led to reduced plasma membrane expression of ANO1, but not AE2, alkalization of intracellular pH and uncontrolled bile acid influx. High intracellular calcium conditions led to annexin A11 membrane shift and colocalization with ANO1. Incubation with IRC patient serum inhibited annexin A11 membrane shift and reduced ANO1 surface expression. CONCLUSION: Cholangiocellular annexin A11 mediates apical membrane abundance of the chloride channel ANO1, thereby supporting biliary bicarbonate secretion. Insertion is inhibited by IRC patient serum containing anti-annexin A11 IgG1/IgG4-autoantibodies. Anti-annexin A11 autoantibodies may contribute to the pathogenesis of IRC by weakening the 'biliary bicarbonate umbrella'. LAY SUMMARY: We previously identified annexin A11 as a specific autoantigen in immunoglobulin G4-related cholangitis (IRC), a B-cell driven disease affecting the bile ducts. Human cholangiocytes are protected against harmful hydrophobic bile acid influx by a defense mechanism referred to as the 'biliary bicarbonate umbrella'. We found that annexin A11 is required for the formation of a robust bicarbonate umbrella. Binding of patient-derived annexin A11 autoantibodies inhibits annexin A11 function, possibly contributing to bile duct damage by weakening the biliary bicarbonate umbrella in patients with IRC.

ATP8B1 Deficiency Results in Elevated Mitochondrial Phosphatidylethanolamine Levels and Increased Mitochondrial Oxidative Phosphorylation in Human Hepatoma Cells.

ATP8B1 is a phospholipid flippase that is deficient in patients with progressive familial intrahepatic cholestasis type 1 (PFIC1). PFIC1 patients suffer from severe liver disease but also present with dyslipidemia, including low plasma cholesterol, of yet unknown etiology. Here we show that ATP8B1 knockdown in HepG2 cells leads to a strong increase in the mitochondrial oxidative phosphorylation (OXPHOS) without a change in glycolysis. The enhanced OXPHOS coincides with elevated low-density lipoprotein receptor protein and increased mitochondrial fragmentation and phosphatidylethanolamine levels. Furthermore, expression of phosphatidylethanolamine N-methyltransferase, an enzyme that catalyzes the conversion of mitochondrial-derived phosphatidylethanolamine to phosphatidylcholine, was reduced in ATP8B1 knockdown cells. We conclude that ATP8B1 deficiency results in elevated mitochondrial PE levels that stimulate mitochondrial OXPHOS. The increased OXPHOS leads to elevated LDLR levels, which provides a possible explanation for the reduced plasma cholesterol levels in PFIC1 disease.

Neutrophils interact with cholangiocytes to cause cholestatic changes in alcoholic hepatitis.

BACKGROUND & OBJECTIVES: Alcoholic hepatitis (AH) is a common but life-threatening disease with limited treatment options. It is thought to result from hepatocellular damage, but the presence of cholestasis worsens prognosis, so we examined whether bile ducts participate in the pathogenesis of this disease. DESIGN: Cholangiocytes derived from human bile ducts were co-cultured with neutrophils from patients with AH or controls. Loss of type 3 inositol 1,4,5-trisphosphate receptor (ITPR3), an apical intracellular calcium channel necessary for cholangiocyte secretion, was used to reflect cholestatic changes. Neutrophils in contact with bile ducts were quantified in liver biopsies from patients with AH and controls and correlated with clinical and pathological findings. RESULTS: Liver biopsies from patients with AH revealed neutrophils in contact with bile ducts, which correlated with biochemical and histological parameters of cholestasis. Cholangiocytes co-cultured with neutrophils lost ITPR3, and neutrophils from patients with AH were more potent than control neutrophils. Biochemical and histological findings were recapitulated in an AH animal model. Loss of ITPR3 was attenuated by neutrophils in which surface membrane proteins were removed. RNA-seq analysis implicated integrin ß1 (ITGB1) in neutrophil-cholangiocyte interactions and interference with ITGB1 on cholangiocytes blocked the ability of neutrophils to reduce cholangiocyte ITPR3 expression. Cell adhesion molecules on neutrophils interacted with ITGB1 to trigger RAC1-induced JNK activation, causing a c-Jun-mediated decrease in ITPR3 in cholangiocytes. CONCLUSIONS: Neutrophils bind to ITGB1 on cholangiocytes to contribute to cholestasis in AH. This previously unrecognised role for cholangiocytes in this disease alters our understanding of its pathogenesis and identifies new therapeutic targets.

Laminin 511-E8, an autoantigen in IgG4-related cholangitis, contributes to cholangiocyte protection.

Background & Aims: IgG4-related cholangitis (IRC) is the hepatobiliary manifestation of IgG4-related disease. Anti-laminin 511-E8 autoantibodies have been identified in its pancreatic manifestation. Laminin 511-E8 promotes endothelial barrier function, lymphocyte recruitment, and cholangiocyte differentiation. Here, we investigate anti-laminin 511-E8 autoantibody presence in IRC, and mechanisms via which laminin 511 may contribute to cholangiocyte protection. Methods: Anti-laminin 511-E8 serum autoantibody positivity was assessed by ELISA. RNA sequencing and RT-qPCR were performed on human H69 cholangiocytes treated with recombinant laminin 511-E8. H69 cholangiocytes were subjected to shRNA knockdown targeting genes encoding laminin 511 (LAMA5, LAMB1, LAMC1) or treated with recombinant laminin 511-E8. Cholangiocellular bile acid influx was quantified radiochemically using 22,23-3H-glycochenodeoxycholic acid (GCDC). GCDC-induced apoptosis was determined by Caspase-3/7 assays. Cholangiocellular barrier function was assessed by FITC-Dextran permeability assays. Immunofluorescent staining of laminin 511 and claudin 1 was performed on extrahepatic bile duct tissue of control and anti-laminin 511-E8 positive individuals with IRC. Results: Seven out of 52 individuals with IRC had autoantibodies against laminin 511-E8. Recombinant laminin 511-E8 led to differential expression of genes involved in secretion, barrier function, and inflammation. Knockdown of laminin 511 constituents increased toxic bile acid permeation and GCDC-induced apoptosis. Laminin 511-E8 treatment decreased toxic bile acid permeation and dose-dependently alleviated GCDC-induced apoptosis. LAMA5 and LAMC1 knockdown increased transepithelial permeability. Laminin 511-E8 treatment reduced transepithelial permeability and prevented T lymphocyte-induced barrier dysfunction. Laminin 511 and claudin 1 staining patterns appeared altered in anti-laminin 511-E8 positive individuals with IRC. Conclusions: Laminin 511-E8 is an autoantigen in subsets of individuals with IRC. Laminin 511 enhances cholangiocellular barrier function and protects cholangiocytes against T lymphocyte-induced barrier dysfunction, toxic bile acid permeation and bile acid-induced apoptosis. Impact and implications: A subset of patients with IgG4-related cholangitis (IRC) has autoantibodies against laminin 511-E8. In human cholangiocytes, laminin 511 protects against (T lymphocyte-induced) epithelial barrier dysfunction and hydrophobic bile acids. Laminin 511 and claudin 1 staining may be altered in extrahepatic bile ducts of patients with IRC who are anti-laminin 511-E8 positive. This makes it tempting to speculate that a decreased epithelial barrier function with attraction of immune cells and impaired bicarbonate secretion as a result of dysfunction of laminin 511 by autoantibody binding could potentially be a common systemic pathogenic mechanism in a subset of patients with IgG4-RD.

Galectin-3 and prohibitin 1 are autoantigens in IgG4-related cholangitis without clear-cut protective effects against toxic bile acids.

Background and aims: IgG4-related cholangitis (IRC) is the hepatobiliary manifestation of IgG4-related disease, a systemic B cell-driven fibro-inflammatory disorder. Four autoantigens have recently been described in IgG4-RD: annexin A11, galectin-3, laminin 511-E8, and prohibitin 1. We have previously reported a protective role of annexin A11 and laminin 511-E8 in human cholangiocytes against toxic bile acids. Here, we explored the potentially protective role of the carbohydrate-binding lectin galectin-3 and the scaffold proteins prohibitins 1 and 2. Methods: Anti-galectin-3, anti-prohibitin 1 and 2 autoantibody positivity in IRC and healthy and disease (primary sclerosing cholangitis (PSC)) control sera was assessed by ELISA/liquid chromatography-tandem mass spectrometry (LC-MS/MS). Human H69 cholangiocytes were subjected to short hairpin RNA (shRNA) knockdown targeting galectin-3 (LGALS3), prohibitin 1 (PHB1), and prohibitin 2 (PHB2). H69 cholangiocytes were also exposed to recombinant galectin-3, the inhibitor GB1107, recombinant prohibitin 1, and the pan-prohibitin inhibitor rocaglamide. Protection against bile acid toxicity was assessed by intracellular pH (pHi) measurements using BCECF-AM, 22,23-3H-glycochenodeoxycholic acid (3H-GCDC) influx, and GCDC-induced apoptosis using Caspase-3/7 assays. Results: Anti-galectin-3 autoantibodies were detected in 13.5% of individuals with IRC but not in PSC. Knockdown of LGALS3 and galectin-3 inhibition with GB1107 did not affect pHi, whereas recombinant galectin-3 incubation lowered pHi. LGALS3 knockdown increased GCDC-influx but not GCDC-induced apoptosis. GB1107 reduced GCDC-influx and GCDC-induced apoptosis. Recombinant galectin-3 tended to decrease GCDC-influx and GCDC-induced apoptosis. Anti-prohibitin 1 autoantibodies were detected in 61.5% and 35.7% of individuals with IRC and PSC, respectively. Knockdown of PHB1, combined PHB1/2 KD, treatment with rocaglamide, and recombinant prohibitin 1 all lowered pHi. Knockdown of PHB1, PHB2, or combined PHB1/2 did not alter GCDC-influx, yet knockdown of PHB1 increased GCDC-induced apoptosis. Conversely, rocaglamide reduced GCDC-influx but did not attenuate GCDC-induced apoptosis. Recombinant prohibitin 1 did not affect GCDC-influx or GCDC-induced apoptosis. Finally, anti-galectin-3 and anti-prohibitin 1 autoantibody pretreatment did not lead to increased GCDC-influx. Conclusions: A subset of individuals with IRC have autoantibodies against galectin-3 and prohibitin 1. Gene-specific knockdown, pharmacological inhibition, and recombinant protein substitution did not clearly disclose a protective role of these autoantigens in human cholangiocytes against toxic bile acids. The involvement of these autoantibodies in processes surpassing epithelial secretion remains to be elucidated.

Role of Bile Acids and the Biliary HCO3- Umbrella in the Pathogenesis of Primary Biliary Cholangitis.

The biliary HCO3- umbrella hypothesis states that human cholangiocytes and hepatocytes create a protective apical alkaline barrier against millimolar concentrations of potentially toxic glycine-conjugated bile salts in bile by secreting HCO3- into the bile duct lumen. This alkaline barrier may retain biliary bile salts in their polar, deprotonated, and membrane-impermeant state to avoid uncontrolled invasion of apolar toxic bile acids, which initiate apoptosis, autophagy and senescence. In primary biliary cholangitis, defects of the biliary HCO3- umbrella, leading to impaired biliary HCO3- secretion have been identified. Current medical therapies stabilize the putatively defective biliary HCO3- umbrella and improve long-term prognosis.

Maternal hypothyroxinaemia in early pregnancy and problem behavior in 5-year-old offspring.

INTRODUCTION: There is evidence, though not consistent, that offspring born to mothers with subtle decreases in thyroid function early in their pregnancies may be at risk of cognitive impairments and attention problems. However, other types of problem behavior have not been addressed thus far. We tested whether maternal thyroid function in early pregnancy is associated with several types of problem behavior in offspring at age 5-6 years. METHODS: This was a longitudinal study that included the data of 2000 mother-child pairs from the Amsterdam Born Children and their Development study. At a median gestational age of 12.9 (interquartile range: 11.9-14.1) weeks, maternal blood was sampled for assessment of free T4 and TSH. Overall problem behavior, hyperactivity/inattention, conduct problems, emotional problems, peer relationship problems and prosocial behavior were measured at age 5-6 years using the Strengths and Difficulties Questionnaire, which was filled out by both parents and teachers. RESULTS: Maternal hypothyroxinaemia <5th percentile was associated with a 1.70 (95% confidence interval (CI): 1.01-2.86) increased odds of teacher-reported hyperactivity/inattention after adjustment for confounders. By increasing the cut-off level to <10th percentile, the odds ratio became 1.47 (95% CI: 0.99-2.20). There were no associations between maternal thyroid function parameters and hyperactivity/inattention as reported by parents, nor with teacher or parent reports of other types of problem behavior. CONCLUSIONS: Our results partially confirm previous observations, showing that early disruptions in the maternal thyroid hormone supply may be associated with ADHD symptoms in offspring. Our study adds that there is no evidence for an effect on other types of problem behavior.