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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. Valeria Rachela Villella2,
  3. Angela Vasaturo2,
  4. Ida Giardino1,
  5. Massimo Pettoello Mantovani1,
  6. Stefano Guido2,
  7. Olivier N Cexus3,
  8. Nick Peake3,
  9. Marco Londei4,
  10. Sonia Quaratino3,
  11. Luigi Maiuri1,*
  1. 1 Institute of Pediatrics, University of Foggia, Italy;
  2. 2 Department of Chemical Engineering, University Federico II of Naples, Italy;
  3. 3 Cancer Research UK Oncology Unit, University of Southampton, United Kingdom;
  4. 4 Novartis Pharma AG Translational Medicine Basel, Switzerland
  1. Correspondence to: LUIGI MAIURI, Pediatrics, University of Foggia, Italy, Istituto di Pediatria Universitaria, Ospedali Riuniti Foggia, via L Pinto 1, FOGGIA, 71100, Italy; maiuri{at}unina.it

Abstract

Background: An unresolved question in Coeliac disease (CD) 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 induced an intracellular pro-oxidative envoronment favoring TG2 activation and leading to the innate immune response.

Methods: Time-course of intracellular delivery to lysosomes of p31-43, pα2 or pα9 gliadin peptides was analyzed 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-MAP-kinase or tyrosine phosphorylation were investigated in cell lines and cultured CD biopsies with/without anti-oxidant treatment or TG2 gene silencing by immunoprecipitation, western blot, confocal microscopy and FRET analysis.

Results: After 24 hours of challenge p31-43, but not pα2 or pα9 , is still retained within LAMP1-positive perinuclear vescicles and leads to increased levels of Reactive Oxygen Species (ROS) that inhibit TG2 ubiquitination and lead to increase 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 restore PPARγ levels and reverse 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 ROS-TG2 axis. TG2 works as a rheostat of ubiquitination and proteasome degradation and drives inflammation via PPARγ downregulation.

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