- Email: [email protected]
Immunological effects of transglutaminase-treated gluten in coeliac disease
Human Immunology 73 (2012) 992–997
Contents lists available at SciVerse ScienceDirect
www.ashi-hla.org
journal homepage: www.elsevier.com/locate/humimm
Immunological effects of transglutaminase-treated gluten in coeliac disease Luca Elli a,b,⇑, Leda Roncoroni a, Martin Hils c, Ralf Pasternack c, Donatella Barisani d, Claudia Terrani b, Valentina Vaira e, Stefano Ferrero e,f, Maria Teresa Bardella a a
Center for Prevention and Diagnosis of Coeliac Disease, Fondazione IRCCS Cà Granda-Ospedale Maggiore Policlinico-Milano, Italy Gastroenterology II, Fondazione IRCCS Cà Granda-Ospedale Maggiore Policlinico-Milano, Italy c Zedira Gmbh, Darmstadt, Germany d Department of Experimental Medicine, University of Milano-Bicocca, Italy e Division of Pathology, Fondazione IRCCS Cà Granda-Ospedale Maggiore Policlinico-Milano, Italy f Department of Biomedical, Surgical and Dental Sciences, Università degli Studi di Milano, Italy b
a r t i c l e
i n f o
Article history: Received 10 December 2011 Accepted 13 July 2012 Available online 23 July 2012
a b s t r a c t 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). Interferonc, 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 c 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 c, 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. Ó 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. 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
Abbreviations: APC, antigen presenting cell; bTG, bacterial transglutaminase; CD, coeliac disease; EmA, anti endomisium antibodies; GFD, gluten free diet; Lys, lysine; HLA, human leukocyte antigen; PT glut, peptic tryptic digested gluten; rh, recombinant human; rhTG2, recombinant human transglutaminase type 2; TG2, transglutaminase type 2; tTGA, anti transglutaminase antibodies. ⇑ Corresponding author. Address: Center for Prevention and Diagnosis of Coeliac Disease, Fondazione IRCCS Cà-Granda Ospedale Maggiore Policlinico-Milano, Via F. Sforza 35, 20122 Milano, Italy. Fax: +39 0250320403. E-mail address: [email protected] (L. Elli).
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 [4–6]. The high
0198-8859/$36.00 - see front matter Ó 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humimm.2012.07.318
L. Elli et al. / Human Immunology 73 (2012) 992–997
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 [9–11]. 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.
993
heating (85 °C for 45 min). Obtained suspension was cooled to room temperature and centrifuged at 17.000 rpm, 20 °C for 45 min in order to remove insoluble compounds. The supernatant was filtered using a 0.22 lm membrane and dialysed at 8 °C in a Spectra/Por 6 dialysis tubing (Molecular Weight Cut-off: 1.000) in 5 L 10 mM NH4HCO3. After 1 h the dialysis tubing was transferred to 5 L fresh NH4HCO3-solution and incubated at 8 °C overnight. Preparation was aliquotated, freezed at 35 °C, and subsequently freeze dried. Enzymatic modifications of gluten were obtained by incubation with bacterial TG (bTG, Activa, Ajinomoto, Tokyo, Japan) (bTG-PTglut) or human recombinant TG2 (rhTG2, Zedira, Darmstadt, Germany) (rhTG2-PT-glut) 10 lg/mL for 16 h at 37 °C in presence of CaCl2 10 mM, with or without lysine (L-lysine 0.5 mg/mL, 15 molar excess) (bTG-Lys-PT-glut or rhTG2-Lys-PT-glut). In control preparations lysine was added alone (Lys-PT-glut). Protein enzymatic result was checked by SDS–PAGE and HPLC analysis. 2.3. Duodenal specimen cultures and treatments
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]. Seventeen CD on a GFD were enrolled and their clinical and demographic characteristics are shown in Table 1.
During EGDS (Olympus endoscopes, Tokyo, Japan), duodenal tissue specimens were taken by the use of disposable endoscopic forceps (Boston Scientific, Boston, USA), for a total of 66 duodenal specimens (3.9 biopsies/patient); after excision, they were rapidly dipped into sterile tubes (Becton and Dickinson, Milano, Italy) containing 2 mL of medium composed by DMEM (GIBCO, Milano, Italy) added with penicillin 100 U/mL-streptomycin 100 lg/mL (GIBCO, Milano, Italy). After 2 washes with the same medium, duodenal specimens were cultured for 48 h at 37 °C in 5% CO2 atmosphere in 2 mL sterile tubes (Eppendorf, Milano, Italy) in the 1 mL of the previously described medium supplemented with 20% fetal calf serum (GIBCO, Milano, Italy). Nine biopsies were exposed to 1 mg/mL of PT-gluten (PT-glut), 20 to transglutaminasetreated gluten (10 to bTG-PT-glut and 10 to rhTG2-PT-glut), 20 to transglutaminase plus lysine-treated gluten (10 to bTG-Lys-PT-glut and 10 to rhTG2-Lys-PT-glut). Nine duodenal specimens without any treatment (CTR) and 8 exposed to Lys-PT-glut were used as controls. At the end of the treatments the medium was taken, aliquoted and rapidly frozen at 80 °C; the bioptic samples were washed 3 times in PBS and formalin-fixed (10%).
2.2. Protein preparations
2.4. Anti endomysium anti tissue transglutaminase IgA antibodies
Wheat flour gluten was a gift from Tate & Lyle (Amygluten, Tate & Lyle, Aalst, Belgium). Gluten (100 mg) was suspended in 100 mL NaOAc, 1 mM EDTA at pH 6.0 (final gluten concentration 1 mg/ mL) and was peptic–tryptic (PT) digested as follows. Gluten suspension was incubated with pepsin 2 mg (Merck, Darmstadt, Germany) for 2 h at 37 °C after having lowered pH to 2 with 1 M HCl acid solution. pH was then adjusted to 7.8 (using 1 M NaOH) and a further 2 h incubation at 37 °C with 2 mg of trypsin (Merck, Darmstadt, Germany) was performed. Enzymatic digestion was inactivated by
Medium release of IgA autoantibodies was measured by means of immunofluorescence (EmA) and ELISA (tTGA) techniques. EmA release was measured following the manufacturer’s instructions (Euroimmun, Lubeck, Germany). Briefly, 20 lL of medium was challenged with a primate esophagus substrate and subsequently exposed to a secondary anti IgA FITC conjugated antibody. Qualitative analysis was performed by a specifically trained biologist (CT). tTGA analysis was performed using of a colorimetric ELISA technique according to manufacturer’s instructions (Eurospital, Trieste, Italy). Ten lL of medium was dispensed in microwells containing TG2 epitopes. After incubation, the antibodies binding with TG2 epitopes was revealed by a biotin/streptavidin and peroxidase technique. Optical density was measured at 450 nm using a microplate reader (Bouty diagnostici, Milan, Italy). All samples were blindly double analyzed, with the addition of positive and negative controls for each analysis run.
2. Methods 2.1. Patients
Table 1 Clinical and demographic characteristics of the celiac patients. Coeliac patients (n = 17) Males Age (mean, range) at endoscopy Age (mean, range) at diagnosis Years at gluten free diet (mean, range) Anti transglutaminase IgAa Marsh modified scale Marsh 0 Marsh 1 Marsh 2 a
At the time of the study.
2 45 (25–73) 37 (22–66) 7 (1–19) All negative 4 8 5
2.5. Transglutaminase, Interferon c and lactate dehydrogenase measurements TG2 activity in the medium was measured by a colorimetric technique (Covalab, Lyon, France) according to manufacturer’s
994
L. Elli et al. / Human Immunology 73 (2012) 992–997
instructions. Fifty microliters of the sample were dispensed in a 96 wells microtiter plate with covalently coupled CBZ-Gln-Gly and incubated with calcium, dithiothereitol and biotinylated cadaverine. After washing, streptavidin-labelled peroxidase was added and peroxidase activity revealed by incubation in presence of H2O2 and tetramethyl benzidine. Optical density was measured at 450 nm using a microplate reader (Bouty diagnostici, Milan, Italy). Interferon c (IFNc) was measured colorimetrically following the manufacturer’s instructions (Endogen, Rockford, USA) by means of ELISA technique. Lactate dehydrogenase (LDH) medium activity was determined photometrically through the conversion of NADH to NAD following the manufacturer’s instructions (Roche, Mannheim, Germany). 2.6. Transglutaminase type 2 immunohistochemistry The presence and distribution of tissue TG2 was evaluated on paraffin-embedded sections as previously described. Antigen retrieval was obtained by 30 min in 95 °C citrate buffer immersion and immunohistochemistry performed using the mouse monoclonal anti TG2 antibody clone TG 100 (Zedira, Darmstadt, Germany) at a dilution 1:5000 using an automated slide stainer (Biogenex, S. Ramon, USA). After incubation with HRP-conjugated secondary antibody, results were visualized using the Envision kit (Dako Cytomation, Glostrup, Denmark). Positive and negative controls were added. 2.7. Mucosal IgA deposits IgA deposits were investigated through a modification of the method previously described by Korponay–Szabo et al. [13] using paraffin-embedded samples supposed to be suitable for immunofluorescence as described by Rantala et al. [14] Paraffin-embedded sample sections were mounted on polylysinated slides (Kaltec, Padova, Italy) and incubated 6 min with Bio-Clear solution (BioOptica, Milano, Italy). After rehydration samples were incubated with a FITC conjugated goat anti human IgA antibody 1:150 (Dako, Milano, Italy) and successively washed once by a 5 min immersion in PBS plus 1% Tween 20 (sigma, Milano, Italy) at room temperature. Slides were analyzed and image acquired by fluorescence microscope (Zeiss, Milano, Italy). Due to the modification from the original method, we check the correctness of IgA deposits through an internal control evaluating the colocalization of the deposits with TG2 by means of double
immunofluorescence technique. Always following the KorponaySzabo method [13], dewaxed, rehydrated and blocked (PBS 3% BSA) slides were incubated overnight at room temperature with primary monoclonal antibody to TG2 (clone CUB 7402, Thermo Scientific, Rockford, USA). Successively, samples were incubated with fluorescein isothiocyanate labelled anti human IgA antibody (Dako, Glostrup, Denmark) and a rhodamine conjugated anti mouse antibody (Dako, Glostrup, Denmark). Slides were mounted with DAPI (Dako, Glostrup, Denmark), analyzed at a fluorescent microscope (Zeiss, Milano, Italy). This check conformed a colocalization of IgA deposits with TG2. 2.8. Statistical analysis Data are reported as mean ± standard deviation (SD) or median and range. Ccomparisons and associations of the data were done by one way ANOVA and chi-squared test. Statistical analyses were performed using the SPSS 13 statistical software (SPSS, Chicago, USA). A p value of 0.05 was regarded as significant. Graphs were designed using Graph Pad Prism 5 software (GraphPad software inc., La Jolla, USA). 3. Results 3.1. 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 bTG-PT-glut or rhTG2-PT-glut (71% in both rhTG2-PT-glut and bTG-PT-glut EmA positive). One hundred percent of the Lys-PTglut treated biopsies resulted positive to EmA test, percentage that dropped to levels comparable to controls when bTG-Lys-PT-glut or rhTG2-Lys-PT-glut were employed, being 28% for both rhTG2-LysPT-glut and bTG-Lys-PT-glut (Fig. 1A). Medium tTGA levels were in accordance with the EmA data; values (U/mL mean ± SD) were 0.4 ± 0.3 in CTR, 1.1 ± 0.5, 1.0 ± 0.5 and 1.0 ± 0.4 in PT-glut, rhTG2-PT-glut and bTG-PT-glut, respectively (p < 0.05 PT-glut or TG treated glut vs. CTR). In rhTG2-Lys-PT-glut, bTG-Lys-PT-glut
Fig. 1. Anti endomisium IgA antibodies (EmA) (A) and anti tissue transglutaminase IgA (B) levels in the medium of cultured duodenal endoscopic biopsies from coeliac patients following a gluten free diet. Biopsies were cultured for 48 h without any treatment (CTR) or exposed to bTG-PT-glut, rhTG2-PT-glut, Lys-PT-glut, bTG-Lys-PT-glut or rhTG2-Lys-PT-glut. ⁄ = p < 0.05 vs. CTR.
L. Elli et al. / Human Immunology 73 (2012) 992–997
995
Fig. 2. Example of a duodenal biopsy containing IgA mucosal deposits, detected by means of immunofluorescence techinique.
and Lys-PT-glut exposed biopsies, tTGA levels were 0.4 ± 0.2, 0.2 ± 0.1 and 0.6 ± 0.3, respectively (Fig. 1B). The release in the medium of EmA was related to the presence of IgA deposits in the cultured biopsy as demonstrated by the immunofluorescence analysis (Fig. 2); in fact all EmA positive specimens presented IgA deposits. On the other hand, the analysis of the specimens obtained from EmA negative samples in the CTR, bTG-Lys-PT-gluten and rhTG2-Lys-PT-gluten group revealed the presence of IgA deposits in 71%, 75% and 75% of the biopsies, respectively. These data suggest that the absence of EmA in the supernatant could be considered a negative result due to a lack of (or partial) activation of the immune response and/or of the antibody release. 3.2. Interferonc, transglutaminase and lactate dehydrogenase activities and transglutaminase type 2 immunohistochemistry Immune activation in ex vivo cultured duodenal biopsies from CD patients was present in samples exposed to PT-glut, rhTG2PT-glut, bTG-PT-glut and Lys-PT-glut as shown by the significantly increased levels of IFNc in the culture medium. In fact, IFNc levels (pg/mL mean ± SD) were 1.3 ± 1.5, 19.5 ± 5.0, 17.7 ± 4.1, 20.5 ± 2.1, 2.1 ± 0.6, 1.1 ± 0.8, 13.5 ± 4.3 in CTR, PT-glut, rhTG2-PT-glut, bTGPT-glut, rhTG2-Lys-PT-glut, bTG-Lys-PT-glut, and Lys-PT-glut, respectively (Fig. 3A). The same trend was observed for TG activity in the medium of cultured biopsies; TG activity (lU/mL mean ± SD) was undetectable in CTR, rhTG2-Lys-PT-glut and bTG-Lys-PT-glut groups, whereas values of 1.3 ± 0.9, 2.3 ± 0.8, 2.1 ± 0.6, 1.9 ± 0.7 were detected in PT-glut, rhTG2-PT-glut, bTG-PT-glut and Lys-PTglut groups, respectively (Fig. 3B). The increase of TG medium activity was related to an augmented TG2 expression in the duodenal mucosa after 48 h of culture as demonstrated by immunohistochemistry in PT-glut, rhTG2-PT-glut, bTG-PT-glut and Lys-PT-glut groups (Fig. 4). The necrotic effect due to the time of culture did not differ according to the treatment, as demonstrated by LDH release in the medium which was not statistically different among the groups (Fig. 3C). Clinical (age at diagnosis, years on gluten free diet) and demographic (sex and age at time of endoscopy) were not statistically associated with any of the parameters tested. 4. 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
Fig. 3. Interferon c (IFNc) levels (A), transglutaminase (TG) activity (B) and Lactate dehydrogenise levels (C) in the medium of cultured duodenal endoscopic biopsies from coeliac patients following a gluten free diet. Biopsies were cultured for 48 h without any treatment (CTR) or exposed to bTG-PT-glut, rhTG2-PT-glut, Lys-PTglut, bTG-Lys-PT-glut or rhTG2-Lys-PT-glut. ⁄ = p < 0.05 vs. CTR; § = below the detectable limit.
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 hyperplasia and increased number of intraepithelial lymphocytes (IELs) and, in general, for the CD syndrome [17]. Pivotal for the development of the pathologic process is the interaction between TG2, gluten and gluten-derived peptides [1,18]. The knowledge about this interaction is extremely important for the development of the so-called peptide-based therapies for CD, which aim to substitute the GFD with chemically or genetically modified glutens or derived peptides [11,19,20]. If the introduction of genetically
996
L. Elli et al. / Human Immunology 73 (2012) 992–997
Fig. 4. Transglutaminase type 2 immunohistochemistry of duodenal endoscopic biopsies from coeliac patients cultured without any treatment (A) or exposed to PT-glut 1 mg/mL (B) for 48 h.
modified plants into the food chain involves ethical problems, costs and long periods of time [21], the enzymatic modification of gluten in order to prevent its immunologic effects on the coeliac intestinal mucosa could represent an accessible strategy. In fact, enzymatic pre-treatment of food it is a diffuse procedure in alimentary industry and, in particular, bTG is widely used for ameliorating food aspect, consistency and thus to make the product more competitive on the market [22]. It has been demonstrated that different gluten-derived peptides are able to stimulate a proinflammatory response driven by peripheral and intestinal CD4+ T cells [23–28]; to exert this effect, peptides should undergo to a TG2-mediated enzymatic process (mainly deamidation) before being exposed on the HLA molecules of the APCs in the duodenal lamina propria [1]. In a severely damaged tissues and in presence of high calcium levels (above 0.5 mM), TG2 forms a covalent thioester intermediate with the distal free amide group of protein-bound glutamines through their active side thiol group, causing the release of an ammonia molecule [29]. The free thiol group of the enzyme interacts with a second nucleophilic acceptor substrate, such as a lysine residue, which then reacts with the peptidylglutamyl–thioester intermediate to form a e(c-glutamyl)lysine or isopeptide bond [30]. Without an acceptor or in condition of low pH (as in inflamed tissue), H2O can act as alternative acceptor, thus causing the deamidation of the donor glutamine to a glutamic acid residue [29]. Gluten-derived peptides are optimal substrates for TG2 because of their high number of glutamine residues, and in a high calcium/low pH environment they can be deamidated to glutamic acid. This modification changes the charge of gluten derived peptides conferring a high affinity for the HLA type II DQ2 molecules [31], fact that triggers the immune response. Given this pathomechanism, insertion of lysine as a nucleophilic acceptor in a TG-mediated process could lead to the interruption of deamidation and, consequently, to stop antigen presentation of deamidated/negatively charged peptides alone or linked to TG2 in an aptenic form, extremely dangerous for the susceptible CD mucosa. This mechanism could be at the basis of our results which demonstrate an inhibition of the immunostimulation after the TG-mediated insertion of lysine into gluten. The inclusion in the diet of modified proteins no longer suitable for antigen presentation, or the use of proteins in which the insertion of side chain molecules changes their solubility or 3D conformation, creating an incompatibility with HLA pockets, appear plausible approaches to develop new CD therapies in the light of our results, the CD pathogenesis and the actual use of TG in food industry [9,11,32]. In the present study we analyzed if an enzymatic modification of gluten by TG could prevent the inflammatory stimulation in an ex vivo model of CD. We treated gluten with rhTG2 or bTG in presence or absence of an acceptor lysine in order to induce a transamidation of the glutamine residues. bTG is a calcium nondependent form of TG that is active on the same sites and sequences of TG2 and it is largely used in the food industry, for its
ability (as human TG) to improve the functional properties of proteins, such as their nutritional value, texture, taste and preservability; bTG is used for pork, beef, poultry, fish and indeed milk products and thus, bTG is largely available and probably ingested in large amounts in the common Western diet [33]. Our data show that modification induced by rhTG2 or bTG alone does not induce a reduction of immunostimulation when compared to unmodified gluten, confirming the results obtained by other researchers on single deamidated peptides [27,28], whereas the addition of lysine to the enzymatic reaction abolishes the release of IFNc and of specific antibodies (EmA and tTGA IgA), as well as the increase of TG activity in the culture medium. This last parameter has been chosen due to the well known induction of TG expression induced by gluten and gliadin in the CD duodenal mucosa. In the present experiments TG activity in the medium was detected only after treatments with immunostimulatory proteins (PT-glut, bTG-PT-glut or rhTG2-PT-glut), but it remained below the sensitivity of the used methodology when duodenal specimens were treated with bTG-Lys-PT-glut or rhTG2-Lys-PTglut, proteins not inducing either an IFNc or an antibodies release. These data are in accordance to what previously reported by Gianfrani et al. [28] that observed a reduced activation of T cell clones obtained from the duodenal mucosa of coeliac patients during incubation with ‘‘lysinated’’ peptides, data that mainly represent the inhibition of the adaptative immune response towards the immunodominant peptides. The data presented in our study were obtained on cultured duodenal biopsies, a model that mimics the physiology of the intestine, since it maintains the normal cell-tocell interaction. The lack of activation of the immune system observed after treatment with bTG-Lys-PT-glut or rhTG2-Lys-PT-glut could also suggest that not only the adaptive immunity, but also other components of the immune system are not activated. Thus, our findings on the duodenal tissue, taken together with Gianfrani’s ones, clearly indicate that insertion of lysine in the gluten protein could lead to alternative strategy to GFD. In conclusion, our results demonstrate that enzymatic pretreatment of gluten with rhTG2 or bTG plus lysine could prevent the duodenal immunostimulation in CD subjects, opening the possibility of the reintroduction of gluten derived food products in CD [34].
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 CàGranda.
L. Elli et al. / Human Immunology 73 (2012) 992–997
References [1] Elli L, Bergamini CM, Bardella MT, Schuppan D. Transglutaminases in inflammation and fibrosis of the gastrointestinal tract and the liver. Dig Liver Dis 2009;41(8):541. [2] D’Arienzo R, Stefanile R, Maurano F, Luongo D, Bergamo P, Mazzarella G, Troncone R, Auricchio S, David C, Rossi M. A deregulated immune response to gliadin causes a decreased villus height in DQ8 transgenic mice. Eur J Immunol 2009;39(12):3552. [3] Shan L, Molberg O, Parrot I, Hausch F, Filiz F, Gray GM, Sollid LM, Khosla C. Structural basis for gluten intolerance in celiac sprue. Science 2002;297(5590):2275. [4] Dieterich W, Ehnis T, Bauer M, Donner P, Volta U, Riecken EO, Schuppan D. Identification of tissue transglutaminase as the autoantigen of celiac disease. Nat Med 1997;3(7):797. [5] Dieterich W, Esslinger B, Schuppan D. Pathomechanisms in celiac disease. Int Arch Allergy Immunol 2003;132(2):98. [6] Fleckenstein B, Molberg O, Qiao SW, Schmid DG, von der Mulbe F, Elgstoen K, Jung G, Sollid LM. Gliadin T cell epitope selection by tissue transglutaminase in celiac disease. Role of enzyme specificity and pH influence on the transamidation versus deamidation process. J Biol Chem 2002;277(37):34109. [7] Berti C, Roncoroni L, Falini ML, Caramanico R, Dolfini E, Bardella MT, Elli L, Terrani C, Forlani F. Celiac-related properties of chemically and enzymatically modified gluten proteins. J Agric Food Chem 2007;55(6):2482. [8] Falini ML, Elli L, Caramanico R, Bardella MT, Terrani C, Roncoroni L, Doneda L, Forlani F. Immunoreactivity of antibodies against transglutaminasedeamidated gliadins in adult celiac disease. Dig Dis Sci 2008;53:2697. [9] Bardella MT, Molteni N, Prampolini L, Giunta AM, Baldassarri AR, Morganti D, Bianchi PA. Need for follow up in coeliac disease. Arch Dis Child 1994;70(3):211. [10] Nachman F, del Campo MP, Gonzalez A, Corzo L, Vazquez H, Sfoggia C, Smecuol E, Sanchez MI, Niveloni S, Sugai E, Maurino E, Bai JC. Long-term deterioration of quality of life in adult patients with celiac disease is associated with treatment noncompliance. Dig Liver Dis 2010;42(10):685. [11] Caputo I, Lepretti M, Martucciello S, Esposito C. Enzymatic strategies to detoxify gluten: implications for celiac disease. Enzyme Res 2010:174354. [12] Oberhuber G, Granditsch G, Vogelsang H. The histopathology of coeliac disease: time for a standardized report scheme for pathologists. Eur J Gastroenterol Hepatol 1999;11(10):1185. [13] Korponay-Szabo IR, Halttunen T, Szalai Z, Laurila K, Kiraly R, Kovacs JB, Fesus L, Maki M. In vivo targeting of intestinal and extraintestinal transglutaminase 2 by coeliac autoantibodies. Gut 2004;53(5):641. [14] Rantala I, Maki M, Laasonen A, Visakorpi JK. Periodate-lysineparaformaldehyde as fixative for the study of duodenal mucosa. Morphologic and immunohistochemical results at light and electron microscopic levels. Acta Pathol Microbiol Immunol Scand A 1985;93(4):165. [15] Tack GJ, Verbeek WH, Schreurs MW, Mulder CJ. The spectrum of celiac disease: epidemiology, clinical aspects and treatment. Nat Rev Gastroenterol Hepatol 2010;7(4):204. [16] Schuppan D, Dennis MD, Kelly CP. Celiac disease: epidemiology, pathogenesis, diagnosis, and nutritional management. Nutr Clin Care 2005;8(2):54. [17] Bardella MT, Velio P, Cesana BM, Prampolini L, Casella G, Di Bella C, Lanzini A, Gambarotti M, Bassotti G, Villanacci V. Coeliac disease: a histological followup study. Histopathology 2007;50(4):465.
997
[18] Dorum S, Arntzen MO, Qiao SW, Holm A, Koehler CJ, Thiede B, Sollid LM, Fleckenstein B: The preferred substrates for transglutaminase 2 in a complex wheat gluten digest are Peptide fragments harboring celiac disease T-cell epitopes. PLoS One 5(11):e14056. [19] Schuppan D, Junker Y, Barisani D. Celiac disease: from pathogenesis to novel therapies. Gastroenterology 2009;137(6):1912. [20] Osorio C, Wen N, Gemini R, Zemetra R, von Wettstein D, Rustgi S. Targeted modification of wheat grain protein to reduce the content of celiac causing epitopes. Funct Integr Genomics 2012. [21] Domingo JL, Gine Bordonaba J. A literature review on the safety assessment of genetically modified plants. Environ Int 2011;37(4):734. [22] Griffin M, Casadio R, Bergamini CM. Transglutaminases: nature’s biological glues. Biochem J 2002;368(Pt 2):377. [23] Molberg O, McAdam S, Lundin KE, Kristiansen C, Arentz-Hansen H, Kett K, Sollid LM. T cells from celiac disease lesions recognize gliadin epitopes deamidated in situ by endogenous tissue transglutaminase. Eur J Immunol 2001;31(5):1317. [24] Sjostrom H, Lundin KE, Molberg O, Korner R, McAdam SN, Anthonsen D, Quarsten H, Noren O, Roepstorff P, Thorsby E, Sollid LM. Identification of a gliadin T-cell epitope in coeliac disease: general importance of gliadin deamidation for intestinal T-cell recognition. Scand J Immunol 1998;48(2):111. [25] Molberg O, McAdam SN, Korner R, Quarsten H, Kristiansen C, Madsen L, Fugger L, Scott H, Noren O, Roepstorff P, Lundin KE, Sjostrom H, Sollid LM. Tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut-derived T cells in celiac disease. Nat Med 1998;4(6):713. [26] Lundin KE, Sollid LM, Anthonsen D, Noren O, Molberg O, Thorsby E, Sjostrom H. Heterogeneous reactivity patterns of HLA-DQ-restricted, small intestinal T-cell clones from patients with celiac disease. Gastroenterology 1997;112(3):752. [27] Maiuri L, Ciacci C, Ricciardelli I, Vacca L, Raia V, Auricchio S, Picard J, Osman M, Quaratino S, Londei M. Association between innate response to gliadin and activation of pathogenic T cells in coeliac disease. Lancet 2003;362(9377):30. [28] Gianfrani C, Siciliano RA, Facchiano AM, Camarca A, Mazzeo MF, Costantini S, Salvati VM, Maurano F, Mazzarella G, Iaquinto G, Bergamo P, Rossi M. Transamidation of wheat flour inhibits the response to gliadin of intestinal T cells in celiac disease. Gastroenterology 2007;133(3):780. [29] Stamnaes J, Fleckenstein B, Sollid LM. The propensity for deamidation and transamidation of peptides by transglutaminase 2 is dependent on substrate affinity and reaction conditions. Biochim Biophys Acta 2008;1784(11):1804. [30] D’Argenio G, Calvani M, Della Valle N, Cosenza V, Di Matteo G, Giorgio P, Margarucci S, Petillo O, Jori FP, Galderisi U, Peluso G. Differential expression of multiple transglutaminases in human colon: impaired keratinocyte transglutaminase expression in ulcerative colitis. Gut 2005;54(4):496. [31] Stenberg P, Roth EB, Sjoberg K. Transglutaminase and the pathogenesis of coeliac disease. Eur J Intern Med 2008;19(2):83. [32] Nachman F, del Campo MP, Gonzalez A, Corzo L, Vazquez H, Sfoggia C, Smecuol E, Sanchez MI, Niveloni S, Sugai E, Maurino E, Bai JC: Long-term deterioration of quality of life in adult patients with celiac disease is associated with treatment noncompliance. Dig Liver Dis 42(10):685. [33] Yokoyama K, Nio N, Kikuchi Y. Properties and applications of microbial transglutaminase. Appl Microbiol Biotechnol 2004;64(4):447. [34] Elli L. Coeliac disease: between ‘‘pizza’’ and ethics. Gut 2006;55(11):1672.
Contents lists available at SciVerse ScienceDirect
www.ashi-hla.org
journal homepage: www.elsevier.com/locate/humimm
Immunological effects of transglutaminase-treated gluten in coeliac disease Luca Elli a,b,⇑, Leda Roncoroni a, Martin Hils c, Ralf Pasternack c, Donatella Barisani d, Claudia Terrani b, Valentina Vaira e, Stefano Ferrero e,f, Maria Teresa Bardella a a
Center for Prevention and Diagnosis of Coeliac Disease, Fondazione IRCCS Cà Granda-Ospedale Maggiore Policlinico-Milano, Italy Gastroenterology II, Fondazione IRCCS Cà Granda-Ospedale Maggiore Policlinico-Milano, Italy c Zedira Gmbh, Darmstadt, Germany d Department of Experimental Medicine, University of Milano-Bicocca, Italy e Division of Pathology, Fondazione IRCCS Cà Granda-Ospedale Maggiore Policlinico-Milano, Italy f Department of Biomedical, Surgical and Dental Sciences, Università degli Studi di Milano, Italy b
a r t i c l e
i n f o
Article history: Received 10 December 2011 Accepted 13 July 2012 Available online 23 July 2012
a b s t r a c t 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). Interferonc, 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 c 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 c, 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. Ó 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. 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
Abbreviations: APC, antigen presenting cell; bTG, bacterial transglutaminase; CD, coeliac disease; EmA, anti endomisium antibodies; GFD, gluten free diet; Lys, lysine; HLA, human leukocyte antigen; PT glut, peptic tryptic digested gluten; rh, recombinant human; rhTG2, recombinant human transglutaminase type 2; TG2, transglutaminase type 2; tTGA, anti transglutaminase antibodies. ⇑ Corresponding author. Address: Center for Prevention and Diagnosis of Coeliac Disease, Fondazione IRCCS Cà-Granda Ospedale Maggiore Policlinico-Milano, Via F. Sforza 35, 20122 Milano, Italy. Fax: +39 0250320403. E-mail address: [email protected] (L. Elli).
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 [4–6]. The high
0198-8859/$36.00 - see front matter Ó 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humimm.2012.07.318
L. Elli et al. / Human Immunology 73 (2012) 992–997
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 [9–11]. 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.
993
heating (85 °C for 45 min). Obtained suspension was cooled to room temperature and centrifuged at 17.000 rpm, 20 °C for 45 min in order to remove insoluble compounds. The supernatant was filtered using a 0.22 lm membrane and dialysed at 8 °C in a Spectra/Por 6 dialysis tubing (Molecular Weight Cut-off: 1.000) in 5 L 10 mM NH4HCO3. After 1 h the dialysis tubing was transferred to 5 L fresh NH4HCO3-solution and incubated at 8 °C overnight. Preparation was aliquotated, freezed at 35 °C, and subsequently freeze dried. Enzymatic modifications of gluten were obtained by incubation with bacterial TG (bTG, Activa, Ajinomoto, Tokyo, Japan) (bTG-PTglut) or human recombinant TG2 (rhTG2, Zedira, Darmstadt, Germany) (rhTG2-PT-glut) 10 lg/mL for 16 h at 37 °C in presence of CaCl2 10 mM, with or without lysine (L-lysine 0.5 mg/mL, 15 molar excess) (bTG-Lys-PT-glut or rhTG2-Lys-PT-glut). In control preparations lysine was added alone (Lys-PT-glut). Protein enzymatic result was checked by SDS–PAGE and HPLC analysis. 2.3. Duodenal specimen cultures and treatments
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]. Seventeen CD on a GFD were enrolled and their clinical and demographic characteristics are shown in Table 1.
During EGDS (Olympus endoscopes, Tokyo, Japan), duodenal tissue specimens were taken by the use of disposable endoscopic forceps (Boston Scientific, Boston, USA), for a total of 66 duodenal specimens (3.9 biopsies/patient); after excision, they were rapidly dipped into sterile tubes (Becton and Dickinson, Milano, Italy) containing 2 mL of medium composed by DMEM (GIBCO, Milano, Italy) added with penicillin 100 U/mL-streptomycin 100 lg/mL (GIBCO, Milano, Italy). After 2 washes with the same medium, duodenal specimens were cultured for 48 h at 37 °C in 5% CO2 atmosphere in 2 mL sterile tubes (Eppendorf, Milano, Italy) in the 1 mL of the previously described medium supplemented with 20% fetal calf serum (GIBCO, Milano, Italy). Nine biopsies were exposed to 1 mg/mL of PT-gluten (PT-glut), 20 to transglutaminasetreated gluten (10 to bTG-PT-glut and 10 to rhTG2-PT-glut), 20 to transglutaminase plus lysine-treated gluten (10 to bTG-Lys-PT-glut and 10 to rhTG2-Lys-PT-glut). Nine duodenal specimens without any treatment (CTR) and 8 exposed to Lys-PT-glut were used as controls. At the end of the treatments the medium was taken, aliquoted and rapidly frozen at 80 °C; the bioptic samples were washed 3 times in PBS and formalin-fixed (10%).
2.2. Protein preparations
2.4. Anti endomysium anti tissue transglutaminase IgA antibodies
Wheat flour gluten was a gift from Tate & Lyle (Amygluten, Tate & Lyle, Aalst, Belgium). Gluten (100 mg) was suspended in 100 mL NaOAc, 1 mM EDTA at pH 6.0 (final gluten concentration 1 mg/ mL) and was peptic–tryptic (PT) digested as follows. Gluten suspension was incubated with pepsin 2 mg (Merck, Darmstadt, Germany) for 2 h at 37 °C after having lowered pH to 2 with 1 M HCl acid solution. pH was then adjusted to 7.8 (using 1 M NaOH) and a further 2 h incubation at 37 °C with 2 mg of trypsin (Merck, Darmstadt, Germany) was performed. Enzymatic digestion was inactivated by
Medium release of IgA autoantibodies was measured by means of immunofluorescence (EmA) and ELISA (tTGA) techniques. EmA release was measured following the manufacturer’s instructions (Euroimmun, Lubeck, Germany). Briefly, 20 lL of medium was challenged with a primate esophagus substrate and subsequently exposed to a secondary anti IgA FITC conjugated antibody. Qualitative analysis was performed by a specifically trained biologist (CT). tTGA analysis was performed using of a colorimetric ELISA technique according to manufacturer’s instructions (Eurospital, Trieste, Italy). Ten lL of medium was dispensed in microwells containing TG2 epitopes. After incubation, the antibodies binding with TG2 epitopes was revealed by a biotin/streptavidin and peroxidase technique. Optical density was measured at 450 nm using a microplate reader (Bouty diagnostici, Milan, Italy). All samples were blindly double analyzed, with the addition of positive and negative controls for each analysis run.
2. Methods 2.1. Patients
Table 1 Clinical and demographic characteristics of the celiac patients. Coeliac patients (n = 17) Males Age (mean, range) at endoscopy Age (mean, range) at diagnosis Years at gluten free diet (mean, range) Anti transglutaminase IgAa Marsh modified scale Marsh 0 Marsh 1 Marsh 2 a
At the time of the study.
2 45 (25–73) 37 (22–66) 7 (1–19) All negative 4 8 5
2.5. Transglutaminase, Interferon c and lactate dehydrogenase measurements TG2 activity in the medium was measured by a colorimetric technique (Covalab, Lyon, France) according to manufacturer’s
994
L. Elli et al. / Human Immunology 73 (2012) 992–997
instructions. Fifty microliters of the sample were dispensed in a 96 wells microtiter plate with covalently coupled CBZ-Gln-Gly and incubated with calcium, dithiothereitol and biotinylated cadaverine. After washing, streptavidin-labelled peroxidase was added and peroxidase activity revealed by incubation in presence of H2O2 and tetramethyl benzidine. Optical density was measured at 450 nm using a microplate reader (Bouty diagnostici, Milan, Italy). Interferon c (IFNc) was measured colorimetrically following the manufacturer’s instructions (Endogen, Rockford, USA) by means of ELISA technique. Lactate dehydrogenase (LDH) medium activity was determined photometrically through the conversion of NADH to NAD following the manufacturer’s instructions (Roche, Mannheim, Germany). 2.6. Transglutaminase type 2 immunohistochemistry The presence and distribution of tissue TG2 was evaluated on paraffin-embedded sections as previously described. Antigen retrieval was obtained by 30 min in 95 °C citrate buffer immersion and immunohistochemistry performed using the mouse monoclonal anti TG2 antibody clone TG 100 (Zedira, Darmstadt, Germany) at a dilution 1:5000 using an automated slide stainer (Biogenex, S. Ramon, USA). After incubation with HRP-conjugated secondary antibody, results were visualized using the Envision kit (Dako Cytomation, Glostrup, Denmark). Positive and negative controls were added. 2.7. Mucosal IgA deposits IgA deposits were investigated through a modification of the method previously described by Korponay–Szabo et al. [13] using paraffin-embedded samples supposed to be suitable for immunofluorescence as described by Rantala et al. [14] Paraffin-embedded sample sections were mounted on polylysinated slides (Kaltec, Padova, Italy) and incubated 6 min with Bio-Clear solution (BioOptica, Milano, Italy). After rehydration samples were incubated with a FITC conjugated goat anti human IgA antibody 1:150 (Dako, Milano, Italy) and successively washed once by a 5 min immersion in PBS plus 1% Tween 20 (sigma, Milano, Italy) at room temperature. Slides were analyzed and image acquired by fluorescence microscope (Zeiss, Milano, Italy). Due to the modification from the original method, we check the correctness of IgA deposits through an internal control evaluating the colocalization of the deposits with TG2 by means of double
immunofluorescence technique. Always following the KorponaySzabo method [13], dewaxed, rehydrated and blocked (PBS 3% BSA) slides were incubated overnight at room temperature with primary monoclonal antibody to TG2 (clone CUB 7402, Thermo Scientific, Rockford, USA). Successively, samples were incubated with fluorescein isothiocyanate labelled anti human IgA antibody (Dako, Glostrup, Denmark) and a rhodamine conjugated anti mouse antibody (Dako, Glostrup, Denmark). Slides were mounted with DAPI (Dako, Glostrup, Denmark), analyzed at a fluorescent microscope (Zeiss, Milano, Italy). This check conformed a colocalization of IgA deposits with TG2. 2.8. Statistical analysis Data are reported as mean ± standard deviation (SD) or median and range. Ccomparisons and associations of the data were done by one way ANOVA and chi-squared test. Statistical analyses were performed using the SPSS 13 statistical software (SPSS, Chicago, USA). A p value of 0.05 was regarded as significant. Graphs were designed using Graph Pad Prism 5 software (GraphPad software inc., La Jolla, USA). 3. Results 3.1. 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 bTG-PT-glut or rhTG2-PT-glut (71% in both rhTG2-PT-glut and bTG-PT-glut EmA positive). One hundred percent of the Lys-PTglut treated biopsies resulted positive to EmA test, percentage that dropped to levels comparable to controls when bTG-Lys-PT-glut or rhTG2-Lys-PT-glut were employed, being 28% for both rhTG2-LysPT-glut and bTG-Lys-PT-glut (Fig. 1A). Medium tTGA levels were in accordance with the EmA data; values (U/mL mean ± SD) were 0.4 ± 0.3 in CTR, 1.1 ± 0.5, 1.0 ± 0.5 and 1.0 ± 0.4 in PT-glut, rhTG2-PT-glut and bTG-PT-glut, respectively (p < 0.05 PT-glut or TG treated glut vs. CTR). In rhTG2-Lys-PT-glut, bTG-Lys-PT-glut
Fig. 1. Anti endomisium IgA antibodies (EmA) (A) and anti tissue transglutaminase IgA (B) levels in the medium of cultured duodenal endoscopic biopsies from coeliac patients following a gluten free diet. Biopsies were cultured for 48 h without any treatment (CTR) or exposed to bTG-PT-glut, rhTG2-PT-glut, Lys-PT-glut, bTG-Lys-PT-glut or rhTG2-Lys-PT-glut. ⁄ = p < 0.05 vs. CTR.
L. Elli et al. / Human Immunology 73 (2012) 992–997
995
Fig. 2. Example of a duodenal biopsy containing IgA mucosal deposits, detected by means of immunofluorescence techinique.
and Lys-PT-glut exposed biopsies, tTGA levels were 0.4 ± 0.2, 0.2 ± 0.1 and 0.6 ± 0.3, respectively (Fig. 1B). The release in the medium of EmA was related to the presence of IgA deposits in the cultured biopsy as demonstrated by the immunofluorescence analysis (Fig. 2); in fact all EmA positive specimens presented IgA deposits. On the other hand, the analysis of the specimens obtained from EmA negative samples in the CTR, bTG-Lys-PT-gluten and rhTG2-Lys-PT-gluten group revealed the presence of IgA deposits in 71%, 75% and 75% of the biopsies, respectively. These data suggest that the absence of EmA in the supernatant could be considered a negative result due to a lack of (or partial) activation of the immune response and/or of the antibody release. 3.2. Interferonc, transglutaminase and lactate dehydrogenase activities and transglutaminase type 2 immunohistochemistry Immune activation in ex vivo cultured duodenal biopsies from CD patients was present in samples exposed to PT-glut, rhTG2PT-glut, bTG-PT-glut and Lys-PT-glut as shown by the significantly increased levels of IFNc in the culture medium. In fact, IFNc levels (pg/mL mean ± SD) were 1.3 ± 1.5, 19.5 ± 5.0, 17.7 ± 4.1, 20.5 ± 2.1, 2.1 ± 0.6, 1.1 ± 0.8, 13.5 ± 4.3 in CTR, PT-glut, rhTG2-PT-glut, bTGPT-glut, rhTG2-Lys-PT-glut, bTG-Lys-PT-glut, and Lys-PT-glut, respectively (Fig. 3A). The same trend was observed for TG activity in the medium of cultured biopsies; TG activity (lU/mL mean ± SD) was undetectable in CTR, rhTG2-Lys-PT-glut and bTG-Lys-PT-glut groups, whereas values of 1.3 ± 0.9, 2.3 ± 0.8, 2.1 ± 0.6, 1.9 ± 0.7 were detected in PT-glut, rhTG2-PT-glut, bTG-PT-glut and Lys-PTglut groups, respectively (Fig. 3B). The increase of TG medium activity was related to an augmented TG2 expression in the duodenal mucosa after 48 h of culture as demonstrated by immunohistochemistry in PT-glut, rhTG2-PT-glut, bTG-PT-glut and Lys-PT-glut groups (Fig. 4). The necrotic effect due to the time of culture did not differ according to the treatment, as demonstrated by LDH release in the medium which was not statistically different among the groups (Fig. 3C). Clinical (age at diagnosis, years on gluten free diet) and demographic (sex and age at time of endoscopy) were not statistically associated with any of the parameters tested. 4. 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
Fig. 3. Interferon c (IFNc) levels (A), transglutaminase (TG) activity (B) and Lactate dehydrogenise levels (C) in the medium of cultured duodenal endoscopic biopsies from coeliac patients following a gluten free diet. Biopsies were cultured for 48 h without any treatment (CTR) or exposed to bTG-PT-glut, rhTG2-PT-glut, Lys-PTglut, bTG-Lys-PT-glut or rhTG2-Lys-PT-glut. ⁄ = p < 0.05 vs. CTR; § = below the detectable limit.
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 hyperplasia and increased number of intraepithelial lymphocytes (IELs) and, in general, for the CD syndrome [17]. Pivotal for the development of the pathologic process is the interaction between TG2, gluten and gluten-derived peptides [1,18]. The knowledge about this interaction is extremely important for the development of the so-called peptide-based therapies for CD, which aim to substitute the GFD with chemically or genetically modified glutens or derived peptides [11,19,20]. If the introduction of genetically
996
L. Elli et al. / Human Immunology 73 (2012) 992–997
Fig. 4. Transglutaminase type 2 immunohistochemistry of duodenal endoscopic biopsies from coeliac patients cultured without any treatment (A) or exposed to PT-glut 1 mg/mL (B) for 48 h.
modified plants into the food chain involves ethical problems, costs and long periods of time [21], the enzymatic modification of gluten in order to prevent its immunologic effects on the coeliac intestinal mucosa could represent an accessible strategy. In fact, enzymatic pre-treatment of food it is a diffuse procedure in alimentary industry and, in particular, bTG is widely used for ameliorating food aspect, consistency and thus to make the product more competitive on the market [22]. It has been demonstrated that different gluten-derived peptides are able to stimulate a proinflammatory response driven by peripheral and intestinal CD4+ T cells [23–28]; to exert this effect, peptides should undergo to a TG2-mediated enzymatic process (mainly deamidation) before being exposed on the HLA molecules of the APCs in the duodenal lamina propria [1]. In a severely damaged tissues and in presence of high calcium levels (above 0.5 mM), TG2 forms a covalent thioester intermediate with the distal free amide group of protein-bound glutamines through their active side thiol group, causing the release of an ammonia molecule [29]. The free thiol group of the enzyme interacts with a second nucleophilic acceptor substrate, such as a lysine residue, which then reacts with the peptidylglutamyl–thioester intermediate to form a e(c-glutamyl)lysine or isopeptide bond [30]. Without an acceptor or in condition of low pH (as in inflamed tissue), H2O can act as alternative acceptor, thus causing the deamidation of the donor glutamine to a glutamic acid residue [29]. Gluten-derived peptides are optimal substrates for TG2 because of their high number of glutamine residues, and in a high calcium/low pH environment they can be deamidated to glutamic acid. This modification changes the charge of gluten derived peptides conferring a high affinity for the HLA type II DQ2 molecules [31], fact that triggers the immune response. Given this pathomechanism, insertion of lysine as a nucleophilic acceptor in a TG-mediated process could lead to the interruption of deamidation and, consequently, to stop antigen presentation of deamidated/negatively charged peptides alone or linked to TG2 in an aptenic form, extremely dangerous for the susceptible CD mucosa. This mechanism could be at the basis of our results which demonstrate an inhibition of the immunostimulation after the TG-mediated insertion of lysine into gluten. The inclusion in the diet of modified proteins no longer suitable for antigen presentation, or the use of proteins in which the insertion of side chain molecules changes their solubility or 3D conformation, creating an incompatibility with HLA pockets, appear plausible approaches to develop new CD therapies in the light of our results, the CD pathogenesis and the actual use of TG in food industry [9,11,32]. In the present study we analyzed if an enzymatic modification of gluten by TG could prevent the inflammatory stimulation in an ex vivo model of CD. We treated gluten with rhTG2 or bTG in presence or absence of an acceptor lysine in order to induce a transamidation of the glutamine residues. bTG is a calcium nondependent form of TG that is active on the same sites and sequences of TG2 and it is largely used in the food industry, for its
ability (as human TG) to improve the functional properties of proteins, such as their nutritional value, texture, taste and preservability; bTG is used for pork, beef, poultry, fish and indeed milk products and thus, bTG is largely available and probably ingested in large amounts in the common Western diet [33]. Our data show that modification induced by rhTG2 or bTG alone does not induce a reduction of immunostimulation when compared to unmodified gluten, confirming the results obtained by other researchers on single deamidated peptides [27,28], whereas the addition of lysine to the enzymatic reaction abolishes the release of IFNc and of specific antibodies (EmA and tTGA IgA), as well as the increase of TG activity in the culture medium. This last parameter has been chosen due to the well known induction of TG expression induced by gluten and gliadin in the CD duodenal mucosa. In the present experiments TG activity in the medium was detected only after treatments with immunostimulatory proteins (PT-glut, bTG-PT-glut or rhTG2-PT-glut), but it remained below the sensitivity of the used methodology when duodenal specimens were treated with bTG-Lys-PT-glut or rhTG2-Lys-PTglut, proteins not inducing either an IFNc or an antibodies release. These data are in accordance to what previously reported by Gianfrani et al. [28] that observed a reduced activation of T cell clones obtained from the duodenal mucosa of coeliac patients during incubation with ‘‘lysinated’’ peptides, data that mainly represent the inhibition of the adaptative immune response towards the immunodominant peptides. The data presented in our study were obtained on cultured duodenal biopsies, a model that mimics the physiology of the intestine, since it maintains the normal cell-tocell interaction. The lack of activation of the immune system observed after treatment with bTG-Lys-PT-glut or rhTG2-Lys-PT-glut could also suggest that not only the adaptive immunity, but also other components of the immune system are not activated. Thus, our findings on the duodenal tissue, taken together with Gianfrani’s ones, clearly indicate that insertion of lysine in the gluten protein could lead to alternative strategy to GFD. In conclusion, our results demonstrate that enzymatic pretreatment of gluten with rhTG2 or bTG plus lysine could prevent the duodenal immunostimulation in CD subjects, opening the possibility of the reintroduction of gluten derived food products in CD [34].
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 CàGranda.
L. Elli et al. / Human Immunology 73 (2012) 992–997
References [1] Elli L, Bergamini CM, Bardella MT, Schuppan D. Transglutaminases in inflammation and fibrosis of the gastrointestinal tract and the liver. Dig Liver Dis 2009;41(8):541. [2] D’Arienzo R, Stefanile R, Maurano F, Luongo D, Bergamo P, Mazzarella G, Troncone R, Auricchio S, David C, Rossi M. A deregulated immune response to gliadin causes a decreased villus height in DQ8 transgenic mice. Eur J Immunol 2009;39(12):3552. [3] Shan L, Molberg O, Parrot I, Hausch F, Filiz F, Gray GM, Sollid LM, Khosla C. Structural basis for gluten intolerance in celiac sprue. Science 2002;297(5590):2275. [4] Dieterich W, Ehnis T, Bauer M, Donner P, Volta U, Riecken EO, Schuppan D. Identification of tissue transglutaminase as the autoantigen of celiac disease. Nat Med 1997;3(7):797. [5] Dieterich W, Esslinger B, Schuppan D. Pathomechanisms in celiac disease. Int Arch Allergy Immunol 2003;132(2):98. [6] Fleckenstein B, Molberg O, Qiao SW, Schmid DG, von der Mulbe F, Elgstoen K, Jung G, Sollid LM. Gliadin T cell epitope selection by tissue transglutaminase in celiac disease. Role of enzyme specificity and pH influence on the transamidation versus deamidation process. J Biol Chem 2002;277(37):34109. [7] Berti C, Roncoroni L, Falini ML, Caramanico R, Dolfini E, Bardella MT, Elli L, Terrani C, Forlani F. Celiac-related properties of chemically and enzymatically modified gluten proteins. J Agric Food Chem 2007;55(6):2482. [8] Falini ML, Elli L, Caramanico R, Bardella MT, Terrani C, Roncoroni L, Doneda L, Forlani F. Immunoreactivity of antibodies against transglutaminasedeamidated gliadins in adult celiac disease. Dig Dis Sci 2008;53:2697. [9] Bardella MT, Molteni N, Prampolini L, Giunta AM, Baldassarri AR, Morganti D, Bianchi PA. Need for follow up in coeliac disease. Arch Dis Child 1994;70(3):211. [10] Nachman F, del Campo MP, Gonzalez A, Corzo L, Vazquez H, Sfoggia C, Smecuol E, Sanchez MI, Niveloni S, Sugai E, Maurino E, Bai JC. Long-term deterioration of quality of life in adult patients with celiac disease is associated with treatment noncompliance. Dig Liver Dis 2010;42(10):685. [11] Caputo I, Lepretti M, Martucciello S, Esposito C. Enzymatic strategies to detoxify gluten: implications for celiac disease. Enzyme Res 2010:174354. [12] Oberhuber G, Granditsch G, Vogelsang H. The histopathology of coeliac disease: time for a standardized report scheme for pathologists. Eur J Gastroenterol Hepatol 1999;11(10):1185. [13] Korponay-Szabo IR, Halttunen T, Szalai Z, Laurila K, Kiraly R, Kovacs JB, Fesus L, Maki M. In vivo targeting of intestinal and extraintestinal transglutaminase 2 by coeliac autoantibodies. Gut 2004;53(5):641. [14] Rantala I, Maki M, Laasonen A, Visakorpi JK. Periodate-lysineparaformaldehyde as fixative for the study of duodenal mucosa. Morphologic and immunohistochemical results at light and electron microscopic levels. Acta Pathol Microbiol Immunol Scand A 1985;93(4):165. [15] Tack GJ, Verbeek WH, Schreurs MW, Mulder CJ. The spectrum of celiac disease: epidemiology, clinical aspects and treatment. Nat Rev Gastroenterol Hepatol 2010;7(4):204. [16] Schuppan D, Dennis MD, Kelly CP. Celiac disease: epidemiology, pathogenesis, diagnosis, and nutritional management. Nutr Clin Care 2005;8(2):54. [17] Bardella MT, Velio P, Cesana BM, Prampolini L, Casella G, Di Bella C, Lanzini A, Gambarotti M, Bassotti G, Villanacci V. Coeliac disease: a histological followup study. Histopathology 2007;50(4):465.
997
[18] Dorum S, Arntzen MO, Qiao SW, Holm A, Koehler CJ, Thiede B, Sollid LM, Fleckenstein B: The preferred substrates for transglutaminase 2 in a complex wheat gluten digest are Peptide fragments harboring celiac disease T-cell epitopes. PLoS One 5(11):e14056. [19] Schuppan D, Junker Y, Barisani D. Celiac disease: from pathogenesis to novel therapies. Gastroenterology 2009;137(6):1912. [20] Osorio C, Wen N, Gemini R, Zemetra R, von Wettstein D, Rustgi S. Targeted modification of wheat grain protein to reduce the content of celiac causing epitopes. Funct Integr Genomics 2012. [21] Domingo JL, Gine Bordonaba J. A literature review on the safety assessment of genetically modified plants. Environ Int 2011;37(4):734. [22] Griffin M, Casadio R, Bergamini CM. Transglutaminases: nature’s biological glues. Biochem J 2002;368(Pt 2):377. [23] Molberg O, McAdam S, Lundin KE, Kristiansen C, Arentz-Hansen H, Kett K, Sollid LM. T cells from celiac disease lesions recognize gliadin epitopes deamidated in situ by endogenous tissue transglutaminase. Eur J Immunol 2001;31(5):1317. [24] Sjostrom H, Lundin KE, Molberg O, Korner R, McAdam SN, Anthonsen D, Quarsten H, Noren O, Roepstorff P, Thorsby E, Sollid LM. Identification of a gliadin T-cell epitope in coeliac disease: general importance of gliadin deamidation for intestinal T-cell recognition. Scand J Immunol 1998;48(2):111. [25] Molberg O, McAdam SN, Korner R, Quarsten H, Kristiansen C, Madsen L, Fugger L, Scott H, Noren O, Roepstorff P, Lundin KE, Sjostrom H, Sollid LM. Tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut-derived T cells in celiac disease. Nat Med 1998;4(6):713. [26] Lundin KE, Sollid LM, Anthonsen D, Noren O, Molberg O, Thorsby E, Sjostrom H. Heterogeneous reactivity patterns of HLA-DQ-restricted, small intestinal T-cell clones from patients with celiac disease. Gastroenterology 1997;112(3):752. [27] Maiuri L, Ciacci C, Ricciardelli I, Vacca L, Raia V, Auricchio S, Picard J, Osman M, Quaratino S, Londei M. Association between innate response to gliadin and activation of pathogenic T cells in coeliac disease. Lancet 2003;362(9377):30. [28] Gianfrani C, Siciliano RA, Facchiano AM, Camarca A, Mazzeo MF, Costantini S, Salvati VM, Maurano F, Mazzarella G, Iaquinto G, Bergamo P, Rossi M. Transamidation of wheat flour inhibits the response to gliadin of intestinal T cells in celiac disease. Gastroenterology 2007;133(3):780. [29] Stamnaes J, Fleckenstein B, Sollid LM. The propensity for deamidation and transamidation of peptides by transglutaminase 2 is dependent on substrate affinity and reaction conditions. Biochim Biophys Acta 2008;1784(11):1804. [30] D’Argenio G, Calvani M, Della Valle N, Cosenza V, Di Matteo G, Giorgio P, Margarucci S, Petillo O, Jori FP, Galderisi U, Peluso G. Differential expression of multiple transglutaminases in human colon: impaired keratinocyte transglutaminase expression in ulcerative colitis. Gut 2005;54(4):496. [31] Stenberg P, Roth EB, Sjoberg K. Transglutaminase and the pathogenesis of coeliac disease. Eur J Intern Med 2008;19(2):83. [32] Nachman F, del Campo MP, Gonzalez A, Corzo L, Vazquez H, Sfoggia C, Smecuol E, Sanchez MI, Niveloni S, Sugai E, Maurino E, Bai JC: Long-term deterioration of quality of life in adult patients with celiac disease is associated with treatment noncompliance. Dig Liver Dis 42(10):685. [33] Yokoyama K, Nio N, Kikuchi Y. Properties and applications of microbial transglutaminase. Appl Microbiol Biotechnol 2004;64(4):447. [34] Elli L. Coeliac disease: between ‘‘pizza’’ and ethics. Gut 2006;55(11):1672.