AUTHOR=Kersten Remco , Trampert David C. , Hubers Lowiek M. , Tolenaars Dagmar , Vos Harmjan R. , van de Graaf Stan F. J. , Beuers Ulrich 

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

JOURNAL=Frontiers in Immunology

VOLUME=14

YEAR=2024

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2023.1251134

DOI=10.3389/fimmu.2023.1251134

ISSN=1664-3224

ABSTRACT=<sec><title>Background and aims</title><p>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.</p></sec><sec><title>Methods</title><p>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 (<italic>LGALS3</italic>), prohibitin 1 (<italic>PHB1</italic>), and prohibitin 2 (<italic>PHB2</italic>). 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 (pH<sub>i</sub>) measurements using BCECF-AM, 22,23-<sup>3</sup>H-glycochenodeoxycholic acid (<sup>3</sup>H-GCDC) influx, and GCDC-induced apoptosis using Caspase-3/7 assays.</p></sec><sec><title>Results</title><p>Anti-galectin-3 autoantibodies were detected in 13.5% of individuals with IRC but not in PSC. Knockdown of <italic>LGALS3</italic> and galectin-3 inhibition with GB1107 did not affect pH<sub>i</sub>, whereas recombinant galectin-3 incubation lowered pH<sub>i</sub>. <italic>LGALS3</italic> 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 <italic>PHB1</italic>, combined <italic>PHB1/2</italic> KD, treatment with rocaglamide, and recombinant prohibitin 1 all lowered pH<sub>i</sub>. Knockdown of <italic>PHB1</italic>, <italic>PHB2</italic>, or combined <italic>PHB1/2</italic> did not alter GCDC-influx, yet knockdown of <italic>PHB1</italic> 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.</p></sec><sec><title>Conclusions</title><p>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.</p></sec>