In vitro models for gluten toxicity: relevance for celiac disease pathogenesis and development of novel treatment options.

In genetically predisposed individuals, dietary gluten in wheat, rye and barley triggers celiac disease, a systemic autoimmune disorder hallmarked by an extensive small-bowel mucosal immune response. The current conception of celiac disease pathogenesis is that it involves components of both innate and adaptive immunity whose activation typically leads to small-bowel villous atrophy with crypt hyperplasia. Currently, the only effective treatment for celiac disease is a strict lifelong gluten-free diet excluding all wheat-, rye- and barley-containing food products. During the diet, the clinical symptoms improve and the small-bowel mucosal damage recovers, while re-introduction of gluten into the diet leads to re-appearance of the symptoms and deterioration of the small-bowel mucosal architecture. In view of the restricted nature of the diet, alternative treatment is warranted. Improved understanding of the molecular basis of celiac disease has enabled researchers to suggest other therapeutic approaches. Although there is no animal model reproducing all features of celiac disease, the use of in vitro approaches including a variety of cell lines and the celiac patient small-bowel mucosal biopsy organ culture has generated knowledge about pathogenesis of celiac disease. In these culture systems, gluten induces different effects that can be quantified, thus also enabling studies concerning the efficacy of candidate therapeutic compounds for celiac disease. This review describes the intestinal epithelial cell models, celiac patient T-cell lines and clones, as well as the small-bowel mucosal organ culture methods widely used in studies of celiac disease, and summarizes the major findings obtained with these systems.

Enzymatic detoxification of gluten by germinating wheat proteases: implications for new treatment of celiac disease.

INTRODUCTION: Currently the only treatment for celiac disease is a lifelong gluten-free diet. The diet is, however, often burdensome, and thus new treatment options are warranted. We isolated proteases from germinating wheat grain naturally meant for total digestion of wheat storage proteins and investigated whether these enzymes can diminish toxic effects of gluten in vitro and ex vivo. METHODS: Pepsin and trypsin digested (PT) gliadin was pretreated with proteases from germinating wheat, whereafter the degradation was analyzed by HPLC-MS (high-performance liquid chromatography and mass spectroscopy) and sodium dodecyl sulphate polyacrylamide gel electrophoresis. The toxicity of cleaved PT-gliadin products was assessed in Caco-2 epithelial cells, celiac patient-derived T cells, and in human small intestinal mucosal organ culture biopsies. RESULTS: Proteases from germinating wheat degraded gliadin into small peptide fragments, which, unlike unprocessed PT-gliadin, did not increase epithelial permeability, induce cytoskeletal rearrangement or changes in ZO-1 expression in Caco-2 cells. Pretreated gliadin did not stimulate T cell proliferation in vitro or enhance the production of autoantibodies to culture supernatants and the activation of CD25+ lymphocytes in the organ culture to the same extent as unprocessed PT-gliadin. DISCUSSION: Germinating wheat enzymes reduce the toxicity of wheat gliadin in vitro and ex vivo. Further studies are justified to develop an alternative therapy for celiac disease.

Secretion of celiac disease autoantibodies after in vitro gliadin challenge is dependent on small-bowel mucosal transglutaminase 2-specific IgA deposits.

BACKGROUND: In celiac disease gluten, the disease-inducing toxic component in wheat, induces the secretion of autoantibodies which are targeted against transglutaminase 2 (TG2). These autoantibodies are produced in the small-intestinal mucosa, where they can be found deposited extracellularly below the epithelial basement membrane and around mucosal blood vessels. In addition, during gluten consumption these autoantibodies can also be detected in patients' serum but disappear from the circulation on a gluten-free diet. Interestingly, after adoption of a gluten-free diet the serum autoantibodies disappear from the circulation more rapidly than the small-intestinal mucosal autoantibody deposits. The toxicity of gluten and the secretion of the disease-specific autoantibodies have been widely studied in organ culture of small-intestinal biopsy samples, but results hitherto have been contradictory. Since the mucosal autoantibodies disappear slowly after a gluten-free diet, our aim was to establish whether autoantibody secretion to organ culture supernatants in treated celiac disease patient biopsies is related to the duration of the diet and further to the pre-existence of mucosal TG2-specific IgA deposits in the cultured biopsy samples. RESULTS: In the organ culture system conducted with biopsies derived from treated celiac disease patients, gliadin induced secretion of autoantibodies to culture supernatants, reduced epithelial cell height and increased the density of lamina proprial CD25+ cells. However, these changes could be demonstrated only in biopsies from short-term treated celiac disease patients, where the small-intestinal mucosal TG2-specific IgA autoantibody deposits were still present. Furthermore, in these biopsies autoantibody secretion could be stimulated fully only after a 48-hour gliadin challenge. CONCLUSION: Our results show that studies focusing on the toxic effects of gliadin in the organ culture system should be carried out with biopsy samples from short-term treated celiac disease patients who are likely still to have mucosal IgA deposits present. In addition to providing an explanation for the discrepancies in previous publications, the present study also enables further validation of the organ culture method.