Lysosomal accumulation of gliadin p31-43 peptide induces oxidative stress and tissue transglutaminase-mediated PPARγ downregulation in intestinal epithelial cells and coeliac mucosa

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)g 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 pa-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 ROSeTG2 axis. TG2 works as a rheostat of ubiquitination and proteasome degradation and drives inflammation via PPARγ downregulation.