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

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

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

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.

Original languageEnglish
Article number1251134
JournalFrontiers in Immunology
Volume14
DOIs
Publication statusPublished - 2023

Keywords

  • IgG4-related systemic disease
  • biliary bicarbonate umbrella
  • cholangiopathy
  • cholestasis
  • immune-mediated disease
  • secretory organs

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