A Celiac Cellular Phenotype, with Altered LPP Sub-Cellular Distribution, Is Inducible in Controls by the Toxic Gliadin Peptide P31-43

Is Inducible in Controls by the Toxic Gliadin Peptide P31-43. PLoS ONE 8(11): e79763. doi:10.1371/journal.pone.0079763 A Celiac Cellular Phenotype, with Altered LPP Sub- Cellular Distribution, Is Inducible in Controls by the Toxic Gliadin Peptide P31-43 Merlin Nanayakkara 0 Roberta Kosova 0 Giuliana Lania 0 Marco Sarno 0 Alessandra Gaito 0 Martina Galatola 0 Luigi Greco 0 Marialaura Cuomo 0 Riccardo Troncone 0 Salvatore Auricchio 0 Renata Auricchio 0 Maria Vittoria Barone 0 Sanjoy Bhattacharya, Bascom Palmer Eye Institute, University of Miami School of Medicine, United States of America 0 1 Department of Translational Medical Science, University of Naples Federico II , Naples , Italy , 2 European Laboratory for the Investigation of Food Induced Disease, (ELFID) University of Naples Federico II , Naples , Italy Celiac disease (CD) is a frequent inflammatory intestinal disease, with a genetic background, caused by gliadin-containing food. Undigested gliadin peptides P31-43 and P57-68 induce innate and adaptive T cell-mediated immune responses, respectively. Alterations in the cell shape and actin cytoskeleton are present in celiac enterocytes, and gliadin peptides induce actin rearrangements in both the CD mucosa and cell lines. Cell shape is maintained by the actin cytoskeleton and focal adhesions, sites of membrane attachment to the extracellular matrix. The locus of the human Lipoma Preferred Partner (LPP) gene was identified as strongly associated with CD using genome-wide association studies (GWAS). The LPP protein plays an important role in focal adhesion architecture and acts as a transcription factor in the nucleus. In this study, we examined the hypothesis that a constitutive alteration of the cell shape and the cytoskeleton, involving LPP, occurs in a cell compartment far from the main inflammation site in CD fibroblasts from skin explants. We analyzed the cell shape, actin organization, focal adhesion number, focal adhesion proteins, LPP sub-cellular distribution and adhesion to fibronectin of fibroblasts obtained from CD patients on a Gluten-Free Diet (GFD) and controls, without and with treatment with A-gliadin peptide P31-43. We observed a ''CD cellular phenotype'' in these fibroblasts, characterized by an altered cell shape and actin organization, increased number of focal adhesions, and altered intracellular LPP protein distribution. The treatment of controls fibroblasts with gliadin peptide P31-43 mimics the CD cellular phenotype regarding the cell shape, adhesion capacity, focal adhesion number and LPP sub-cellular distribution, suggesting a close association between these alterations and CD pathogenesis. - Funding: This study was supported by PRIN 2009 (Research Programs of National Interest), code CUP E61J11000300001. 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 characterized by mucosal inflammation due to the Th1 T cell response to certain gliadin peptides (e.g., the 33-mer A-gliadin peptide) [1] and the innate immune response to other gliadin peptides (e.g., A-gliadin peptide P31-43) [23]. Recent data have shown that gliadin peptides, particularly peptide P31-43, act as growth factors for cell lines, such as CaCo-2, and induce the proliferation of celiac crypt enterocytes [46]. Because both the 33-mer peptide containing P57-68 [78] and the 25-mer peptide containing P31-43 (P31-55) [9] are resistant to hydrolysis by gastric, pancreatic and intestinal proteases, these peptides remain active in vivo in the intestine after gluten ingestion. Alterations of the actin cytoskeleton of epithelial cells are also observed in the celiac mucosa [10], and gluten-dependent shorter enterocyte heights have been described in CD [11]. Tight junction proteins are also altered in active CD, leading to increased permeability [1213]. Furthermore, gliadin and gliadin peptides, including P31-43, interfere with actin rearrangements in both the CD mucosa and cell lines. The in vivo treatment of the small intestine of celiac disease patients with gliadin peptides causes the rearrangement of the actin cytoskeleton in enterocytes [1415]. This phenomenon is also detectable in Caco-2 cells, a cell line commonly used to study gliadin effects on metabolism, barrier functions and apoptosis [16 17]. The treatment of CaCo-2 cells with peptic-tryptic gliadin digest and the truncated toxic peptide P3143 causes rearrangements of the actin cytoskeleton similar to those induced by epidermal growth factor (EGF); [4]. Quantitative microscopic analysis revealed that gliadin directly damages F-actin and tight junction proteins in Lovo cells [18]. The direct interaction of gliadin peptides with actin impairs protein trafficking in COS1 cells [19]. Moreover, gliadin peptides induce cytoskeleton remodeling with altered motility in dendritic cells [20]. CD has been strongly associated with HLA-DQ 2/8 isotypes, which are necessary but not sufficient to produce this autoimmune disease. A total of 39 non-HLA susceptibility loci have recently been identified through repeated GWAS and explain approximately 1014% of the CD heritability; combined with the HLA locus, only 50% of the CD heritability is explained [21]. The strongest non-HLA association signal maps to a 70-kb linkage disequilibrium (LD) block in intron 2 of the LPP gene [22]. LPP protein localizes at focal adhesions, which are sites of membrane attachment to the extracellular matrix, in cell-cell contacts, and in the cytoplasm and nucleus [23]. LPP protein is a proline-rich protein [24] previously identified in fibroblasts [25] that plays an important role in focal adhesion architecture as a versatile scaffolding and adaptor protein and also acts as a transcription factor in the nucleus [23]. As a scaffold and adaptor protein, LPP harbors various binding sites, one of which is a VASP (vasodilator-stimulated phosphoprotein)-binding site, which increases actin polymerization, leading to cell protrusions. After binding to LPP, VASP is recruited to cell adhesion sites, thus directing changes in actin dynamics [23]. The aim of the study was to analyze the actin cytoskeleton, cell shape, focal adhesions and adhesion to the substrate of skin fibroblasts, from CD patients and controls along with the subcellular distribution of the LPP protein. Finally we studied the effects of gliadin peptide P31-43 on the shape and adhesion of controls and CD fibroblasts. Skin fibroblasts, a cell compartment far from the main inflammation site, allowed to investigate differences at base line and following gliadin peptides stimulation in CD patients and controls. Materials and Methods Cell culture and treatments Fibroblasts were cultured from skin biopsies obtained from CD patients and controls. We obtained fibroblasts (...truncated)