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Lysosomal accumulation of gliadin p31–43 peptide induces oxidative stress and tissue transglutaminase-mediated PPARγ downregulation in intestinal epithelial cells and coeliac mucosa
  1. Alessandro Luciani1,2,
  2. Valeria Rachela Villella3,
  3. Angela Vasaturo3,
  4. Ida Giardino4,
  5. Massimo Pettoello-Mantovani1,
  6. Stefano Guido2,3,
  7. Olivier N Cexus5,
  8. Nick Peake5,
  9. Marco Londei6,
  10. Sonia Quaratino5,
  11. Luigi Maiuri1
  1. 1Institute of Pediatrics, University of Foggia, Foggia, Italy
  2. 2Dynamic Imaging Microscopy, CEINGE, Naples, Italy
  3. 3Department of Chemical Engineering, University Federico II of Naples, Naples, Italy
  4. 4Department of Biomedical Science, University of Foggia, Foggia, Italy
  5. 5Cancer Research UK Oncology Unit, University of Southampton, Southampton, UK
  6. 6Novartis Pharma AG Translational Medicine, Basel, Switzerland
  1. Correspondence to Professor Luigi Maiuri, Institute of Pediatrics, University of Foggia, viale Pinto 1, Foggia 71100, Italy; maiuri{at}unina.it

Abstract

Background An unresolved question in coeliac disease is to understand how some toxic gliadin peptides, in particular p31–43, can initiate an innate response and lead to tissue transglutaminase (TG2) upregulation in coeliac intestine and gliadin sensitive epithelial cell lines.

Aim We addressed whether the epithelial uptake of p31–43 induces an intracellular pro-oxidative envoronment favouring TG2 activation and leading to the innate immune response.

Methods The time course of intracellular delivery to lysosomes of p31–43, pα-2 or pα-9 gliadin peptides was analysed in T84 and Caco-2 epithelial cells. The effects of peptide challenge on oxidative stress, TG2 and peroxisome proliferator-activated receptor (PPAR)γ ubiquitination and p42/44–mitogen activated protein (MAP) kinase or tyrosine phosphorylation were investigated in cell lines and cultured coeliac disease biopsies with/without anti-oxidant treatment or TG2 gene silencing by immunoprecipitation, western blot, confocal microscopy and Fluorenscence Transfer Resonance Energy (FRET) analysis.

Results After 24 h of challenge p31–43, but not pα-2 or pα-9, is still retained within LAMP1-positive perinuclear vesicles and leads to increased levels of reactive oxygen species (ROS) that inhibit TG2 ubiquitination and lead to increases of TG2 protein levels and activation. TG2 induces cross-linking, ubiquitination and proteasome degradation of PPARγ. Treatment with the antioxidant EUK-134 as well as TG2 gene silencing restored PPARγ levels and reversed all monitored signs of innate activation, as indicated by the dramatic reduction of tyrosine and p42/p44 phosphorylation.

Conclusion p31–43 accumulation in lysosomes leads to epithelial activation via the ROS–TG2 axis. TG2 works as a rheostat of ubiquitination and proteasome degradation and drives inflammation via PPARγ downregulation.

  • Coeliac disease
  • ROS
  • lysosomes
  • tissue transglutaminase
  • PPARγ
  • gastrointestinal immune response
  • immune response
  • inflammatory mechanisms
  • mucosal immunity
  • CD
  • coeliac disease
  • IFN-γ
  • interferon γ
  • TG2
  • tissue transglutaminase
  • PPARγ
  • peroxisome proliferator-activated receptor gamma, ROS, reactive oxygen species, GSH, glutathione
  • MnSOD
  • manganese superoxide dismutase

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Footnotes

  • Linked articles 169656, 189332.

  • Funding This work was supported by the Coeliac UK and the Rothschild Trust Corporation and Associazione Italiana Celiachia Regione Puglia (# 1400/07, Del. Reg.502 – 08/04/2008).

  • Competing interests None.

  • Ethics approval This study was conducted with the approval of the Regione Campania Health Authority.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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