Selective inhibition of the gliadin-specific, cell-mediated immune response by transamidation with microbial transglutaminase

Abstract

CD is an immune-mediated enteropathy caused by the ingestion of wheat gluten. The modification of gluten by intestinal tTGase plays a crucial role in CD pathogenesis. In this study, we observed that extensive transamidation of wheat flour with K-C2H5 by mTGase yielded spf and K-gliadins fractions. By Western blot, we found that these modifications were associated with strongly reduced immune cross-reactivity. With the use of DQ8 tg mice as a model of gluten sensitivity, we observed a dramatic reduction in IFNγ production in gliadin-specific spleen cells challenged with spf and K-gliadins in vitro (n=12; median values: 813 vs. 29 and 99; control vs. spf and K-gliadins, P=0.012 for spf, and P=0.003 for K-gliadins). For spf, we also observed an increase in the IL-10/IFNγ protein ratio (n=12; median values: 0.3 vs. 4.7; control vs. spf, P=0.005). In intestinal biopsies from CD patients challenged in vitro with gliadins (n=10), we demonstrated further that K-gliadins dramatically reduced the levels of antigen-specific IFNγ mRNA in all specimens responsive to native gliadins (four of 10; P<0.05). As cytotoxic effects have been described for gliadins, we also studied GST and caspase-3 activities using the enterocytic Caco-2 cell line. We found that neither activities were modified by flour transamidation. Our results indicate that K-C2H5 cross-linking via mTGase specifically affects gliadin immunogenicity, reversing the inducible inflammatory response in models of gluten sensitivity without affecting other aspects of the biological activity of gliadins.

Biochemical modifications of gliadins induced by microbial transglutaminase on wheat flour

“…Western blot analysis with anti-native gliadin antiserum showed the complete loss of immune reactivity of K-gliadins and spf ( Fig. 2A), in agreement with previous findings [12]. On the contrary, anti-K-gliadin antibodies identified epitopes in all examined samples.…”

“…The production of isopeptide bonds by means of the catalytic activity of mTGase dramatically decreased the gliadin yield in wheat flour to 17.4 ± 0.7% (mean ± SD; Table 1), in agreement with previous results [12]. Interestingly, glutenins were also partially recovered following transamidation treatment (47.4 ± 2.5%, Table 1).…”

“…In particular, gliadin yield decreased to 40% during the first enzymatic step, whereas glutenins resulted to be more markedly reduced during the second transamidation step. In line with these results, the watersoluble protein fraction (spf) [12] sharply increased during the first enzymatic step and peaked at the end of this step (Fig. 1A, lower panel).…”

“…Most importantly, we found that the transamidation of gliadin following the treatment of wheat flour with mTGase and lysine methyl ester (K-CH 3 ) caused a dramatic down-regulation in IFNγ production in vitro in the intestinal T cells of CD patients [11]. Very recently, we demonstrated that wheat flour, following transamidation using a new "two-step" procedure, was selectively associated with positive changes in the phenotype of the antigen-specific immune response in models of gluten sensitivity [12].…”

Vaccination and Other Antigen-Specific Immunomodulatory Strategies in Celiac Disease

“…In silico analysis of a prototypic dominant gliadin peptide, p56-68, suggested that this occurs because binding of the cognate transamidated gliadin peptide to the DQ2 molecule is strongly reduced, even if it is not completely abolished [45]. Furthermore, in DQ8 transgenic mice, the enzymatic modification of whole gliadin was associated with an alteration of the gliadin-inducible IL-10/IFN-γ ratio, indicative of reversal of the phenotype from inflammatory to anti-inflammatory [46]. Therefore, we speculated that the transamidation of dominant peptides might favor HLA binding of low-affinity, subdominant or cryptic, gliadin determinants with dramatic consequences for the immune outcome.…”