Immunological effects of transglutaminase-treated gluten 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.
Section snippets
Patients
Enrolled patients signed an informed consent before the esophagogastroduodenoscopy (EGDS) and the study was approved by the ethical committee of the “Fondazione IRCCS Cà Granda-Ospedale Maggiore Policlinico-Milano”. CD diagnosis was based on the presence of the serological markers anti-tissue-transglutaminase (tTGA, ELISA or radioimmunoassay tests) and/or anti-endomisium (EmA, immunofluorescence technique) IgA antibodies and compatible histology (grade III marsh-Oberhuber classification) [12].
Release of anti endomysium and anti transglutaminase IgA antibodies and mucosal IgA deposits
Release in the medium of specific coeliac-related antibodies (EmA and tTGA) from the ex vivo cultured duodenal mucosa of CD patients was analyzed as marker of B-lymphocytes activation, and is reported in Fig. 1. PT-glut exposition induced a significant increase in EmA and tTGA (Fig. 1A and 1B). EmA were detected in the medium in 86% of PT-glut exposed biopsies as compared to 33% of biopsies incubated with medium alone (p < 0.05) (Fig. 1). The same effect was observed in biopsies exposed to
Discussion
This study demonstrates that the enzymatic treatment of gluten with bTG or rhTG2 plus lysine is able to suppress its immunologic effects on the duodenal mucosa of CD patients.
CD pathogenesis implies a complex network of mechanisms in which genetic and environmental factors cooperate in triggering and fuelling an altered immune response against the self antigen TG2 [15], [16]; this chronic inflammatory state is responsible for the typical duodenal lesion characterized by villous atrophy, crypt
Acknowledgments
The authors have no conflict of interest to declare and they approved the final version of the manuscript. All the authors have participated to the study; in particular LE, LR, CT and DB have been involved in ex vivo cultures, VV and SF performed immunofluorescence, MH and RP prepared proteins and MTB prepared the manuscript. Financial support: Zedira GmbH and Fondazione IRCCS Cà-Granda.
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Microbial transglutaminase: A biotechnological tool to manage gluten intolerance
2020, Analytical BiochemistryManagement of celiac disease in daily clinical practice
2019, European Journal of Internal MedicineCitation Excerpt :In the last years, new therapies have been tested in vitro or in in vivo models of CD with different potential therapeutic targets. These include the proteolytic degradation of gluten peptides, inhibition of intestinal permeability to prevent gluten uptake, the inhibition of TG2, or modulation of the immune response to gluten preventing T cells activation [11,121–123]. The most promising new therapy in CD is therapeutic vaccine, Nexvax2, an adjuvant-free mix of three peptides that include immunodominant epitopes for gluten-specific CD4-positive T cells.
Microbial transglutaminase: A new potential player in celiac disease
2019, Clinical ImmunologyCitation Excerpt :Those statements contradict what is known in the literature. mTg is allergenic [88,98,99], immunogenic [21,26,70,71,100], the mTg treated wheat/gluten products are immunoreactive [21,25,70,90–93,101–104], and its safety is doubtful. Concerning safety, as per the producers, it was checked only in vitro in cell-lines, on animal models like mice, guinea pigs and rats [96] and not on normal or gluten depended human conditions.
Next-generation therapies for celiac disease: The gluten-targeted approaches
2018, Trends in Food Science and TechnologyCitation Excerpt :It should be noted that the main aim of this technology is to avoid the painful symptoms and prevent damage to the small intestine from accidental gluten ingestion, not to allow unrestrained gluten ingestion. An alternative strategy for blocking the immunogenicity of dietary gluten relies on the transamidation of glutamine residues present in gluten proteins with microbial transglutaminase (Brzozowski, 2016; Watanabe et al., 1994) in the presence of specific amine nucleophiles such as l-lysine, l-lysine methyl or ethyl esters prior to ingestion (Elli et al., 2012; Gianfrani et al., 2007; Heredia-Sandoval, Islas-Rubio, Cabrera-Chavez, & Calderon de la Barca, 2014; Lombardi et al., 2013; Mazzarella et al., 2012; Mazzeo et al., 2013). Thus, the recognition of the glutamine residues and their deamidation by TG2 are blocked, tackling key steps in the pathogenesis of celiac disease (Di Sabatino et al., 2012).
The industrial food additive, microbial transglutaminase, mimics tissue transglutaminase and is immunogenic in celiac disease patients
2016, Autoimmunity ReviewsCitation Excerpt :Additionally, mTg deamidated gluten peptides are recognized by gluten-specific T cells, thus enhancing the immunogenicity of gluten [33]. Most recently, it was shown that mTg-treated gluten peptides applied to cultured intestinal biopsies from CD patients, induced a 15-fold increase in INFγ release, and 2.5- and 2.1-fold increases in medium tTg antibody levels and endomysial antibody positivity, respectively [34]. It can be concluded that both mTg-neo and mTg-modified gluten proteins are immunogenic in the celiac population.
Microbial transglutaminase has a lower deamidation preference than human tissue transglutaminase on a celiac disease relevant wheat gliadin T-cell epitope
2016, Journal of Cereal ScienceCitation Excerpt :Lombardi et al. (2013) showed that prolamin extracted from wheat flour modified with lysine ethyl ester by MTG leads to a significantly reduced immunogenicity compared to unmodified prolamin, which was evaluated by measuring the expression of interferon-gamma and interleukin-10 in stimulated intestinal lymphocytes derived from celiac disease patients. Similar results were obtained after stimulation of duodenal biopsies from CD patients treated with gluten modified with lysine by MTG and TG2 (Elli et al., 2012). Based on these contradictory reports, the effect of MTG treatment in the context of celiac disease remains an open question.