Immunological effects of transglutaminase-treated gluten in coeliac disease

Abstract

Coeliac disease pathogenesis is characterized by an immune response triggered, in genetically predisposed subjects, by ingested gluten and its withdrawal from the diet is the only available therapy. However, enzymatic modification of gluten through the insertion of lysine to avoid antigen presentation could represent a new therapeutical approach for patients.

Sixty-six duodenal biopsies from 17 coeliac patients were cultured for 48 h with gluten or enzymatically-modified gluten (treated with human recombinant transglutaminase type 2 or bacterial transglutaminase, with or without lysine). Interferonγ, anti endomisium and anti transglutaminase IgA antibodies, lactate dehydrogenase and transglutaminase activity were measured in the culture medium. Transglutaminase type 2 expression was evaluated on biopsies by immunohistochemistry.

Gluten and transglutaminase-treated gluten increased by 13–15 fold interferon γ release, as well as antibodies, transglutaminase activity, and the immunohistochemical expression of transglutaminase type 2. Addition of lysine to the enzymatic modification of gluten normalized interferon γ, antibodies, transglutaminase activity and immunohistochemical expression of transglutaminase type 2. Lactate dehydrogenase did not differ among the studied groups.

Enzymatic modification of gluten by transglutaminase plus lysine prevents the immunologic effects on cultured duodenal biopsies from coeliac patients and could be tested as an alternative therapy in coeliac disease.

Introduction

Coeliac disease (CD) is the most frequent chronic enteropathy in Western countries. Its pathogenesis is multifactorial and genetic (HLA type II molecules) and environmental (gluten) factors cooperate in generating a deregulated immune response which in turn causes duodenal lesions, characterized by villous atrophy, intraepithelial lymphocytosis and crypt hyperplasia. Some steps of the CD pathogenesis are still obscure, but it seems clear that only in subjects carrying the HLA type II DQ2 and/or DQ8 haplotypes, the ingestion of peptides from wheat flour can induce an aberrant Th1-driven immune response which eventually turns toward an autoimmune reaction having as target the enzyme transglutaminase type 2 (TG2, also named tissue transglutaminase, tTG) [1], [2]. Crucial for the CD4+ T cell expansion is the presentation of small gluten peptide fragments on the HLA molecules of antigen presenting cells (APCs) [3]. These peptides share similar amino acid sequences, rich in glutamine and proline, and contain the motive Proline–Glutamine–X (different amino acid residues)–Proline–Tyrosine (or Phenylalanine). The high proline content preserves peptides from the enzymatic digestion in the intestinal lumen, whereas the high percentage of glutamine residues (>30%) and the particular tertiary structure make them good substrates for TG2. It has been hypothesized that in the lamina propria of the duodenal mucosa TG2 deamidates gluten peptides, changing their electric charge from neutral to negative and making them suitable for the HLA type II DQ2/DQ8 pocket on the APCs [4], [5], [6]. The high affinity of anti gliadin antibodies for the deamidated forms of gluten is in agreement with this mechanism [7], [8]. Could gluten be modified in order to lose its affinity for HLA molecules, thus blocking the “coeliac cascade”, patients may have an alternative to the only available therapy, i.e. the gluten free diet (GFD). GFD is safe and efficient in most patients; however, due to the large use of gluten in the Western diet, it has an important impact on CD patients’ quality of life, mainly on their social habits and on the variety of dietetic possibilities [9], [10], [11]. The enzymatic pre-treatment and modification of gluten could, theoretically, modify the protein structure of its peptides and prevent or at least reduce the immunological activation observed in CD patients, thus widening dietary options. This goal could be reached by the insertion of lysine in a TG2 mediated reaction with gluten; in fact, if a covalent thioester intermediate between TG2 and the distal free amide group of protein-bound glutamine residue starts the enzymatic reaction, successively, a lysine residue could act as a nucleophilic acceptor substrate with the formation of an isopeptide bond, preventing the affinity of gluten proteins with HLA molecules.

In the present study, we investigated the immunological effects of different transglutaminase-mediated modifications of gluten (with or without the addition of lysine) on an ex vivo model of CD, in order to assess their ability to reduce or eliminate the stimulatory effect of gluten on the immunological response.