Tailoring the immune response to wheat gliadin by enzymatic transamidation.

Enzymatic transamidation of wheat gliadin by microbial transglutaminase inhibits IFN-γ secretion by intestinal T cell lines from celiac disease (CD) patients. Here, we analysed its effects on intestinal biopsies from CD patients and studied the underlying mechanisms in HLA-DQ8 transgenic (tg) mice, a model of T-cell mediated gluten sensitivity. In vitro challenge with a soluble form of transamidated gliadin (spf) upregulated IL-10 transcript levels in human biopsy samples. Furthermore, the ratio of IL-10/IFN-γ transcripts was significantly increased following treatment with spf. In DQ8 tg mice, recall responses in vitro in the presence of dendritic cells pulsed with transamidated gliadin showed that gliadin-specific CD4+ T cells did not produce IFN-γ at any tested dose. On the contrary, spf-specific CD4+ T cells still secreted IFN-γ, but they also produced significant levels of IL-10 with both native and transamidated gliadin. Interestingly, this anti-inflammatory activity was restricted to a specific reverse-phase high-pressure liquid chromatography (RP-HPLC) fraction encompassing α-gliadins. These findings suggested an ability of transamidated gliadin to revert, as well as to prevent, the inflammatory phenotype triggered by native gliadin. This property was intrinsically associated with specific components of the α-gliadin fraction.

Mucosal immunity induced by gliadin-presenting spores of Bacillus subtilis in HLA-DQ8-transgenic mice.

The induction of mucosal immunity requires efficient antigen delivery and adjuvant systems. Probiotic bacterial strains are considered to be very promising tools to address both of these needs. In particular, Bacillus subtilis spores are currently under investigation as a long-lived, protease-resistant adjuvant system for different antigens. Furthermore, a non-recombinant approach has been developed based on the stable adsorption of antigen on the spore surface. In the present study, we explored this strategy as a means of modulating the immune response to wheat gliadin, the triggering agent of celiac disease (CD), an enteropathy driven by inflammatory CD4(+) T cells. Gliadin adsorption was tested on untreated or autoclaved wild-type (wt) and mutant (cotH or cotE) spores. We found that gliadin was stably and maximally adsorbed by autoclaved wt spores. We then tested the immune properties of the spore-adsorbed gliadin in HLA-DQ8-transgenic mice, which express one of the two HLA heterodimers associated with CD. In vitro, spore-adsorbed gliadin was efficiently taken up by mouse dendritic cells (DCs). Interestingly, gliadin-pulsed DCs efficiently stimulated splenic CD4(+) T cells from mice immunised with spore-adsorbed gliadin. Nasal pre-dosing with spore-adsorbed gliadin failed to down-regulate the ongoing cellular response in gliadin-sensitised DQ8 mice. Notably, naïve mice inoculated intranasally with multiple doses of spore-adsorbed gliadin developed an intestinal antigen-specific CD4(+) T cell-mediated response. In conclusion, our data highlight the ability of spore-adsorbed gliadin to elicit a T-cell response in the gut that could be exploitable for developing immune strategies in CD.

Biochemical modifications of gliadins induced by microbial transglutaminase on wheat flour.

BACKGROUND: Celiac disease (CD) is an immune-mediated disorder caused by the ingestion of wheat gluten. A lifelong, gluten-free diet is required to normalize the intestinal mucosa. We previously found that transamidation by microbial transglutaminase (mTGase) suppressed the gliadin-specific immune response in intestinal T-cell lines from CD patients and in models of gluten sensitivity. METHODS: SDS-PAGE, Western blot, ELISA, tissue transglutaminase (tTGase) assay and nano-HPLC-ESI-MS/MS experiments were used to analyze prolamins isolated from treated wheat flour. RESULTS: Gliadin and glutenin yields decreased to 7.6±0.5% and 7.5±0.3%, respectively, after a two-step transamidation reaction that produced a water-soluble protein fraction (spf). SDS-PAGE, Western blot and ELISA analyses confirmed the loss of immune cross-reactivity with anti-native gliadin antibodies in residual transamidated gliadins (K-gliadins) and spf as well as the occurrence of neo-epitopes. Nano-HPLC-ESI-MS/MS experiments identified some native and transamidated forms of celiacogenic peptides including p31-49 and confirmed that mTGase had similar stereo-specificity of tTGase. Those peptides resulted to be 100% and 57% modified in spf and K-gliadins, respectively. In particular, following transamidation p31-49 lost its ability to increase tTGase activity in Caco-2 cells. Finally, bread manufactured with transamidated flour had only minor changes in baking characteristics. CONCLUSIONS: The two-step transamidation reaction modified the analyzed gliadin peptides, which are known to trigger CD, without influencing main technological properties. GENERAL SIGNIFICANCE: Our data shed further light on a detoxification strategy alternative to the gluten free diet and may have important implications for the management of CD patients.