Gene Expression Profiling of Gliadin Effects on Intestinal Epithelial Cells Suggests Novel Non-Enzymatic Functions of Pepsin and Trypsin

et al. (2013) Gene Expression Profiling of Gliadin Effects on Intestinal Epithelial Cells Suggests Novel Non-Enzymatic Functions of Pepsin and Trypsin. PLoS ONE 8(6): e66307. doi:10.1371/journal.pone.0066307 Gene Expression Profiling of Gliadin Effects on Intestinal Epithelial Cells Suggests Novel Non-Enzymatic Functions of Pepsin and Trypsin Amarjit Parmar 0 Dario Greco 0 Jarkko Vena la inen 0 Massimiliano Gentile 0 Emma Dukes 0 Pa ivi Saavalainen 0 Philip C. Trackman, Boston University Goldman School of Dental Medicine, United States of America 0 1 Research Programs Unit, Immunobiology, and Haartman Institute, Department of Medical Genetics, University of Helsinki , Helsinki , Finland , 2 Department of Bioscience and Nutrition, Karolinska Institutet, Stockholm, Sweden, 3 Department of Pharmacology and Toxicology, University of Eastern Finland , Kuopio , Finland , 4 Genome Informatics Unit, University of Helsinki , Helsinki , Finland Gliadin triggers T-cell mediated immunity in celiac disease, and has cytotoxic effects on enterocytes mediated through obscure mechanisms. In addition, gliadin transport mechanisms, potential cell surface receptors and gliadin-activated downstream signaling pathways are not completely understood. In order to screen for novel downstream gliadin target genes we performed a systematic whole genome expression study on intestinal epithelial cells. Undifferentiated Caco-2 cells were exposed to pepsin- and trypsin- digested gliadin (PT-G), a blank pepsin-trypsin control (PT) and to a synthetic peptide corresponding to gliadin p31-43 peptide for six hours. RNA from four different experiments was used for hybridization on Agilent one color human whole genome DNA microarray chips. The microarray data were analyzed using the Bioconductor package LIMMA. Genes with nominal p,0.01 were considered statistically significant. Compared to the untreated cells 1705, 1755 and 211 probes were affected by PT-G, PT and p31-43 respectively. 46 probes were significantly different between PT and PT-G treated cells. Among the p31-43 peptide affected probes, 10 and 21 probes were affected by PT-G and PT respectively. Only PT-G affected genes could be validated by quantitative real-time polymerase chain reaction. All the genes were, nonetheless, also affected to a comparable level by PT treated negative controls. In conclusion, we could not replicate previously reported direct effects of gliadin peptides on enterocytes. The results rather suggest that certain epitopes derived from pepsin and trypsin may also affect epithelial cell gene transcription. Our study suggests novel nonenzymatic effects of pepsin and trypsin on cells and calls for proper controls in pepsin and trypsin digested gliadin experiments. It is conceivable that gliadin effects on enterocytes are secondary mediated through oxidative stress, NFkB activation and IL-15 up-regulation. - Funding: This work was funded by the European Union Commission by a Marie Curie Excellence Grant (FP6 contract MEXT-CT-2005-025270)and by the Academy of Finland. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Celiac disease (CD) is an autoimmune disorder caused by the ingestion of gluten present in wheat, barley and rye. It is the alcohol soluble gliadin fraction in gluten which triggers CD in genetically susceptible individuals, i.e. who are HLA-DQ2 or DQ8 positive [13]. The screen detected prevalence of CD is approximately 1% in Caucasians and so far the only cure is a strict adherence to gluten-free diet (GFD) [4,5]. The two hallmarks of CD are villous atrophy and crypt-cell hyperplasia in the small intestine and individuals suffering from CD display a wide range of symptoms, where diarrhoea and malabsorption are only a few to mention [68]. The proteolytic cleavage of gliadin by digestive enzymes in the stomach and intestine produces an array of distinct but overlapping peptides [9]. Some of these peptides are termed immunodominant, as they can activate the adaptive immune system [9]. Pathogenesis in CD is driven by T-cells which recognize selected immunodominant, deamidated gliadin peptides, such as p57-68, presented by the disease associated HLADQ2 or DQ8 molecules on antigen presenting cells [1012]. The T cell activation and mucosal inflammation persists as long as gluten is present in the lamina propria beneath the epithelial layer. Thus, the presence of gluten in the mucosa is a prerequisite for the activation of gluten-reactive T cells and the ensuing inflammation. Another subset of gliadin-derived peptides is termed cytotoxic and these peptides activate the innate immune system which precedes the activation of adaptive immune system in the lamina propria [9,11,13,14]. The gliadin-derived cytotoxic peptides affect the gut in different ways. In experiments on cultured epithelial cells, gliadin peptides have been shown to induce oxidative stress, rearrangement of actin cytoskeleton and impairment of epithelial tight-junction assembly [1417]. Studies by Barone et al. showed that gliadin peptides interfere with endocytic vesicle maturation and also promote cell proliferation by prolonging epidermal growth factor receptor (EGFR) activation [1820]. Furthermore, some toxic gliadin peptides (e.g. p31-43 or p31-49 peptides of gliadin) have been reported to induce apoptosis in gut epithelial cells [21-23]. These effects are thought to be direct effects of gliadin peptides. Interestingly, the immunodominant peptides (e.g. p57-68) have been shown in these studies to be unable to mimic the effects that the cytotoxic peptides (e.g. p31-43 or p31-49) have on enterocytes. Gliadin peptides must cross the epithelial barrier and enter the mucosa to activate T cells. Several mechanisms (paracellular and transcellular) of gliadin transport across the intestinal epithelial barrier have been suggested [19,2427]. In addition, some gliadin peptides have been shown to bind to the chemokine receptor CXCR3 on the surface of epithelial cells and induce tight junction permeability [27]. Enterocyte apoptosis caused by gliadin peptides is also considered a potential mechanism of gliadin introduction into the mucosa. The intestinal epithelial barrier is impaired in CD and as luminal antigens are present in close proximity to enterocytes, an interaction between the two is feasible. Despite a multitude of documented effects attributed to cytotoxic gliadin peptides, the molecular mechanisms through which these effects are brought about remain obscure. No study has thus far been undertaken to study the changes in enterocyte gene expression as a result of their interaction with gliadin peptides. A transcription profile of epithelial cells exposed to gliadin may not only help us identify certain receptor-associated signalling pathways but may also help us understand other gliadin (...truncated)