Modulatory Effect of Gliadin Peptide 10-mer on Epithelial Intestinal CACO-2 Cell Inflammatory Response

et al. (2013) Modulatory Effect of Gliadin Peptide 10-mer on Epithelial Intestinal CACO-2 Cell Inflammatory Response. PLoS ONE 8(6): e66561. doi:10.1371/journal.pone.0066561 Modulatory Effect of Gliadin Peptide 10-mer on Epithelial Intestinal CACO-2 Cell Inflammatory Response Antonella Capozzi 0 Olimpia Vincentini 0 Pietro Gizzi 0 Alessandra Porzia 0 Agostina Longo 0 Cristina Felli 0 Vincenzo Mattei 0 Fabrizio Mainiero 0 Marco Silano 0 Maurizio Sorice 0 Roberta Misasi 0 Salvatore V. Pizzo, Duke University Medical Center, United States of America 0 1 Department of Experimental Medicine, Sapienza University , Rome , Italy , 2 Unit of Human Nutrition and Health, Istituto Superiore di Sanita` , Rome , Italy , 3 Experimental Medicine and Environmental Pathology Laboratory, ''Sapienza'' University , Rieti , Italy Celiac Disease (CD) is a chronic inflammatory enteropathy, triggered in genetically susceptible individuals by dietary gluten. Gluten is able to elicit proliferation of specific T cells and secretion of inflammatory cytokines in the small intestine. In this study we investigated the possibility that p10-mer, a decapeptide from durum wheat (QQPQDAVQPF), which was previously shown to prevent the activation of celiac peripheral lymphocytes, may exert an inhibitory effect on peptic-tryptic digested gliadin (PT-Gly)-stimulated intestinal carcinoma CACO-2 cells. In these cells, incubated with PT-Gly or p31-43 a-gliadin derived peptide in the presence or in the absence of p10-mer, IRAK1 activation and NF-kB, ERK1/2 and p38 MAPK phosphorylation were measured by immunoblotting, Cyclooxigenase 2 (COX-2) activity by PGE-2 release assay, and production of cytokines in the cell supernatants by ELISA. Our results showed that pre-treatment of CACO-2 cells with p10mer significantly inhibited IRAK1 activation and NF-kB, ERK1/2 and p38 MAPK phosphorylation, as well as COX-2 activity (i.e. PGE-2 release) and production of the IL-6 and IL-8 pro-inflammatory cytokines, induced by gliadin peptides. These findings demonstrate the inhibitory effect of the p10-mer peptide on inflammatory response in CACO-2 cells. The results of the present study show that this p10-mer peptide can modulate "in vitro" the inflammatory response induced by gliadin peptides, allowing to move towards new therapeutic strategies. Turning off the inflammatory response, may in fact represent a key target in the immunotherapy of celiac disease. - Funding: This work has been supported by grants from the University La Sapienza School of Medicine (to RM). 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. . These authors contributed equally to this work. Celiac disease (CD) is a chronic inflammatory enteropathy with an autoimmune pathogenesis, triggered in genetically susceptible individuals by dietary gluten, the alcohol protein fraction of cereals, such as wheat, rye and barley. Gliadin is the main protein from wheat gluten. CD is histologically characterized by villous atrophy, crypt cell hyperplasia and increased number of intraepithelial lymphocytes. In addition, patients with celiac disease typically develop antibodies to gliadin and autoantibodies specific for endogenous enzyme transglutaminase 2 (TG2) [1]. At present, many gluten peptides have been described to elicit proliferation and secretion of cytokines, such as interferon-c, of gluten specific T cells in the intestinal mucosa of CD patients [2,3]. Susceptibility to celiac disease is strongly associated with major histocompatibility complex (MHC) class II molecules, HLA-DQ2 and HLA-DQ8. In particular, the immune response, directed against specific gluten antigens, leads to destruction of intestinal epithelial cells (IECs). Although gluten is known to play a role in activating glutenspecific T cells in the lamina propria, recent evidences support the notion that gluten affects the innate immune response [46]. Thus, both innate and adaptive immune responses are necessary to trigger the gluten-dependent mucosal inflammation [7]. Indeed, the a-gliadin-derived peptide p3143 is able to activate the innate immune response in celiac mucosa. Moreover, the activation of the mucosal innate immunity by p3143 has been demonstrated to be required to trigger the immunogenicity of p33-mer, another agliadin derived peptide [810]. Activation of intestinal epithelial cells plays a pivotal role in CD pathogenesis [8] mediating gut inflammation. Intestinal epithelial cells represent an important component of innate immunity and activate sophisticated responses to inflammatory stimuli. The manner in which intestinal epithelial cell polarity affects responses to inflammatory stimuli is largely unknown, but it is clear that intestinal epithelial cells actively participate in directing an inflammatory response [11]. Although the vast majority of bacterial and food components do not elicit intestinal inflammation, it is known that pathogens that cause acute inflammation do activate the NF-kB pathway, resulting in a regulation of genes encoding proinflammatory cytokines, chemokines and adhesion molecules [11,12]. At present, the only known treatment of CD is the life-long withdrawl of gluten containing food from the diet. We described p10-mer, a decapeptide (sequence QQPQDAVQPF) [13] that prevents the activation of peripheral celiac lymphocytes by gliadin peptides [14] and promotes a shift from a Th1-type response toward a Th2-type response in celiac disease [15]. In the present study we evaluated "in vitro" the modulating effect of p10-mer on gliadin-triggered intestinal epithelial cell inflammation. Our results indicated first that this peptide is able to prevent IRAK1 and NF-kB activation, induced by gliadin peptides, in intestinal CACO-2 cells. Materials and Methods Cell culture CACO-2/TC7 cells [16] were cultured in Dulbeccos Modified Medium (high glucose) supplemented with 1% nonessential amino acids and containing 10% fetal calf serum (FCS), 2 mM Lglutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 1 mM Hepes (Invitrogen, Carlsbad, CA, USA) at 37uC in a humified 5% CO2 atmosphere. The culture medium was replaced three times a week. Subculture was performed at 80% of confluence. All experiments were performed between passages 65 and 78. Gliadin preparation and peptic-triptic digestion The peptic-tryptic digest of gliadin (PT-Gly) is a useful tool to study the biological effects of gliadin. The PT-Gly contains some gliadin peptides that show different toxic activity in CD patients tissues. The alcohol-soluble protein fraction from whole cereal flour of bread wheat (Triticum aestivum, variety S Pastore) was extracted and subjected to peptic-tryptic digestion, as previously described [14,17]. Briefly, gliadin was separated from wheat flour by a 70% ethanol aqueous solution. Then, gliadin was exposed at 3 (...truncated)