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

Background and aimsIgG4-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.MethodsAnti-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.ResultsAnti-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.ConclusionsA 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

On the role of IgG4 in inflammatory conditions: lessons for IgG4-related disease

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 Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Janse

Hepatobiliary acid-base homeostasis: Insights from analogous secretory epithelia

Many epithelia secrete bicarbonate-rich fluid to generate flow, alter viscosity, control pH and potentially protect luminal and intracellular structures from chemical stress. Bicarbonate is a key component of human bile and impaired biliary bicarbonate secretion is associated with liver damage. Major efforts have been undertaken to gain insight into acid-base homeostasis in cholangiocytes and more can be learned from analogous secretory epithelia. Extrahepatic examples include salivary and pancreatic duct cells, duodenocytes, airway and renal epithelial cells. The cellular machinery involved in acid-base homeostasis includes carbonic anhydrase enzymes, transporters of the solute carrier family, and intra- and extracellular pH sensors. This pH-regulatory system is orchestrated by protein-protein interactions, the establishment of an electrochemical gradient across the plasma membrane and bicarbonate sensing of the intra- and extracellular compartment. In this review, we discuss conserved principles identified in analogous secretory epithelia in the light of current knowledge on cholangiocyte physiology. We present a framework for cholangiocellular acid-base homeostasis supported by expression analysis of publicly available single-cell RNA sequencing datasets from human cholangiocytes, which provide insights into the molecular basis of pH homeostasis and dysregulation in the biliary system

DataSheet_1_Galectin-3 and prohibitin 1 are autoantigens in IgG4-related cholangitis without clear-cut protective effects against toxic bile acids.pdf

Background and aimsIgG4-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.MethodsAnti-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.ResultsAnti-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.ConclusionsA 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.</p