Diversité de la réponse IgE dans l'allergie à l'arachide : caractérisation des allergènes et devenir de leur potentiel allergénique lors des traitements thermiques et des processus digestifs

L’allergie à l’arachide représente un tiers des allergies alimentaires en France. Elle entraîne des réactions souvent sévères et ne s’atténue que rarement avec l’âge. Elle relève principalement d’une réaction d’hypersensibilité immédiate médiée par les immunoglobulines de type E (IgE) spécifiques de certaines protéines de l’arachide. De nombreuses protéines et fragments peptidiques extraits de la graine, purifiés par chromatographies et caractérisés par séquençage ainsi que par analyse protéomique, ont pu être classés dans trois familles de protéines de réserve des plantes, les globulines 7S, 11S et les albumines 2S. L’étude de la capacité de liaison aux IgE de ces protéines et des peptides qui en dérivent a été menée à l’aide de 80 sérums de patients allergiques à l’arachide par des techniques de type ELISA. Des allergènes majeurs (entraînant une réponse IgE spécifique intense chez la majorité des patients) se retrouvent dans chacune des familles. Les albumines 2S présentent le potentiel allergénique le plus marqué. Chaque patient est sensibilisé à plusieurs protéines. De plus, pour chaque allergène, les IgE peuvent reconnaître plusieurs régions intramoléculaires. Selon les protéines, les différents traitements thermiques peuvent modifier ou non le potentiel allergénique. Les processus digestifs réalisés in vivo et dans des tests de digestibilité in vitro dégradent complètement les protéines mais ne suppriment pas le potentiel allergénique de l’arachide. Les fragments peptidiques de faible masse moléculaire tels que ceux issus de ces hydrolyses enzymatiques jouent vraisemblablement un rôle important dans la mise en place et le développement de l’allergie à l’arachide.\ud ________________________________________________________________________________________________________\ud Peanut allergy represents about 30% of food allergies in France. It is one of the most frequent cause of severe food-associated anaphylaxis and it tends to persist throughout life. Allergy to peanut is an IgE-mediated reaction. Several proteins and peptide fragments derived therefrom were extracted from the kernels. They were purified by a combinaison of selective precipitations and chromatographic separations, then characterized by N-terminal amino acid sequencing and proteomics. They belong to 3 major plant storage protein families, i.e. the 7S and 11S globulins and 2S albumins. The analysis of the IgE-binding capacity of those proteins and derived peptides was performed by Enzyme AllergoSorbent Test (EAST) and EAST inhibition using the sera of 80 peanut allergic patients. The major allergens which lead to an intense specific IgE response in the vast majority of patients were found in each plant protein family. 2S albumins exhibited the most important allergenic potential. Each patient appeared to be sensitized to numerous proteins. Moreover, for each allergen, several intramolecular regions were recognized by specific IgE. Depending on the allergen, the different thermal processings either modified or did not modify the allergenic potential. Proteins were extensively degraded during in vivo and in vitro digestion but their allergenic potential was not abolished. Low molecular weight peptide fragments such as those formed by pepsin and pepsin, trypsin and chymotrypin hydrolysis certainly play a central role in the sensitization process and the development of peanut allergy

Epicutaneous Immunotherapy (EPIT) Blocks the Allergic Esophago-Gastro-Enteropathy Induced by Sustained Oral Exposure to Peanuts in Sensitized Mice

Background: Food allergy may affect the gastrointestinal tract and eosinophilia is often associated with allergic gastrointestinal disorders. Allergy to peanuts is a life-threatening condition and effective and safe treatments still need to be developed. The present study aimed to evaluate the effects of sustained oral exposure to peanuts on the esophageal and jejunal mucosa in sensitized mice. We also evaluated the effects of desensitization with epicutaneous immunotherapy (EPIT) on these processes. Methods: Mice were sensitized by gavages with whole peanut protein extract (PPE) given with cholera toxin. Sensitized mice were subsequently exposed to peanuts via a specific regimen and were then analysed for eosinophilia in the esophagus and gut. We also assessed mRNA expression in the esophagus, antibody levels, and peripheral T-cell response. The effects of EPIT were tested when intercalated with sensitization and sustained oral peanut exposure. Results: Sustained oral exposure to peanuts in sensitized mice led to severe esophageal eosinophilia and intestinal villus sub-atrophia, i.e. significantly increased influx of eosinophils into the esophageal mucosa (136 eosinophils/mm2) and reduced villus/crypt ratios (1.660.15). In the sera, specific IgE levels significantly increased as did secretion of Th2 cytokines by peanut-reactivated splenocytes. EPIT of sensitized mice significantly reduced Th2 immunological response (IgE response and splenocyte secretion of Th2 cytokines) as well as esophageal eosinophilia (50 eosinophils/mm2, p,0.05), mRNA expression of Th2 cytokines in tissue - eotaxin (p,0.05), IL-5 (p,0.05), and IL-13 (p,0.05) -, GATA-3 (p,0.05), and intestinal villus sub-atrophia (2.360.15). EPIT also increased specific IgG2a (p,0.05) and mRNA expression of Foxp3 (p,0.05) in the esophageal mucosa. Conclusions: Gastro-intestinal lesions induced by sustained oral exposure in sensitized mice are efficaciously treated by allergen specific EPIT

Antigen Uptake by Langerhans Cells Is Required for the Induction of Regulatory T Cells and the Acquisition of Tolerance During Epicutaneous Immunotherapy in OVA-Sensitized Mice

The skin is a major immunologic organ that may induce protection, sensitization or tolerance. Epicutaneous immunotherapy (EPIT) has been proposed as an attractive strategy to actively treat food allergy and has been shown to induce tolerance in sensitized mice through the induction of Foxp3+ regulatory T cells (Tregs), especially CD62L+ Tregs. Among immune cells in the skin, dendritic cells are key players in antigen-specific immune activation or regulation. The role of different populations of skin DCs in tolerance induction remains to be elucidated. Using OVA-sensitized BALB/c mice, we demonstrated that the application of a patch containing OVA-A647 to the skin resulted in allergen uptake by Langerhans cells (LCs) and CD11b+ dermal cDC2 and subsequent migration into skin draining lymph nodes. These 2 populations induced Foxp3 expression in CD4+ cells in vitro. Only LCs induced LAP+ cells and CD62L+ Tregs. Using Langerin-eGFP-DTR mice, we analyzed the role of LCs in the mechanisms of tolerance induction by EPIT in vivo. Following complete depletion of LCs, a dramatic decrease in the number of OVA+ DCs and OVA+ CD11b+ dermal cDC2 was observed in skin draining lymph nodes 48 h after epicutaneous application. Likewise, 2 weeks of EPIT in non-depleted mice induced Foxp3+ Tregs, especially CD62L+, and LAP+ Tregs in skin draining lymph nodes and spleen, whereas no induction of Tregs was observed in LC-depleted mice. Following 8 weeks of treatment, EPIT-treated mice showed significant protection against anaphylaxis accompanied by a significant increase of Foxp3+ Tregs, especially CD62L+ Tregs, which was not seen in the absence of LCs. In summary, although both LCs and CD11b+ dermal cDC2s could induce regulatory T cells, the absence of LCs during EPIT impaired treatment efficacy, indicating their crucial role in skin-induced tolerance

Effect of simulated gastro-duodenal digestion on the allergenic reactivity of beta-lactoglobulin

<p>Abstract</p> <p>Background</p> <p>Cow's milk (CM) allergy affects about 2% of infants. The allergenicity of dietary proteins, including those from CM, has been related to their digestibility although the generality of the link and its causality remains to be demonstrated. In this study we use an in vitro digestion system, to investigate the digestibility of β-lactoglobulin (blg) during gastrointestinal transit and to assess the impact of this process on blg allergenic reactivity in CM allergic children.</p> <p>Methods</p> <p>Blg digesta were prepared using an <it>in vitro </it>digestion protocol simulating either gastric digestion alone or followed by duodenal digestion with or without phosphatidylcholine (PC). Biochemical analysis of blg digesta was performed by SDS-PAGE and their concentration was measured by a sandwich ELISA. Assessment of their allergenic reactivity was done <it>in vitro </it>by EAST inhibition, specific basophil activation (basotest) and lymphocyte proliferation (PCNA-flow cytometry) assays using sera and cells from patients allergic to blg and <it>in vivo </it>by skin prick testing (SPT) of these patients.</p> <p>Results</p> <p>Blg was only broken down to smaller peptides after gastro-duodenal digestion although a sizeable amount of intact protein still remained. Digestion did not modify the IgE binding capacity of blg except for gastro-duodenal digestion performed in the absence of PC. These results are consistent with the quantity of intact blg remaining in the digesta. Overall both gastric and gastroduodenal digestion enhanced activation of sensitized basophils and proliferation of sensitized lymphocytes by blg. However, there was a tendency towards reduction in mean diameter of SPT following digestion, the PC alone during phase 1 digestion causing a significant increase in mean diameter.</p> <p>Conclusions</p> <p>Digestion did not reduce the allergenic reactivity of blg to a clinically insignificant extent, PC inhibiting digestion and thereby protecting blg allergenic reactivity. SPT reactivity was reduced compared to blg immunoreactivity in <it>in vitro </it>tests.</p

Diversité de la réponse IgE dans l'allergie à l' arachide (caractérisation des allergènes et devenir de leur potentiel allergénique lors des traitements thermiques et des processus digestifs)

L'allergie à l'arachide représente un tiers des allergies alimentaires en France. Elle entraîne des réactions souvent sévères et ne s'atténue que rarement avec l'âge. Elle relève principalement d'une réaction d'hypersensibilité immédiate médiée par les immunoglobulines de type E (IgE) spécifiques de certaines protéines de l'arachide. De nombreuses protéines et fragments peptidiques extraits de la graine, purifiés par chromatographies et caractérisés par séquençage ainsi que par analyse protéomique, ont pu être classés dans trois familles de protéines de réserve des plantes, les globulines 7S, 11S et les albumines 2S. L'étude de la capacité de liaison aux IgE de ces protéines et des peptides qui en dérivent a été menée à l'aide de 80 sérums de patients allergiques à l'arachide par des techniques de type ELISA. Des allergènes majeurs (entraînant une réponse IgE spécifique intense chez la majorité des patients) se retrouvent dans chacune des familles. Les albumines 2S présentent le potentiel allergénique le plus marqué. Chaque patient est sensibilisé à plusieurs protéines. De plus, pour chaque allergène, les IgE peuvent reconnaître plusieurs régions intramoléculaires. Selon les protéines, les différents traitements thermiques peuvent modifier ou non le potentiel allergénique. Les processus digestifs réalisés in vivo et dans des tests de digestibilité in vitro dégradent complètement les protéines mais ne suppriment pas le potentiel allergénique de l'arachide. Les fragments peptidiques de faible masse moléculaire tels que ceux issus de ces hydrolyses enzymatiques jouent vraisemblablement un rôle important dans la mise en place et le développement de l'allergie à l'arachidePeanut allergy represents about 30% of food allergies in France. It is one of the most frequent cause of severe food-associated anaphylaxis and it tends to persist throughout life. Allergy to peanut is an IgE-mediated reaction. Several proteins and peptide fragments derived therefrom were extracted from the kernels. They were purified by a combinaison of selective precipitations and chromatographic separations, then characterized by N-terminal amino acid sequencing and proteomics. They belong to 3 major plant storage protein families, i.e. the 7S and 11S globulins and 2S albumins. The analysis of the IgE-binding capacity of those proteins and derived peptides was performed by Enzyme AllergoSorbent Test (EAST) and EAST inhibition using the sera of 80 peanut allergic patients. The major allergens which lead to an intense specific IgE response in the vast majority of patients were found in each plant protein family. 2S albumins exhibited the most important allergenic potential. Each patient appeared to be sensitized to numerous proteins. Moreover, for each allergen, several intramolecular regions were recognized by specific IgE. Depending on the allergen, the different thermal processings either modified or did not modify the allergenic potential. Proteins were extensively degraded during in vivo and in vitro digestion but their allergenic potential was not abolished. Low molecular weight peptide fragments such as those formed by pepsin and pepsin, trypsin and chymotrypin hydrolysis certainly play a central role in the sensitization process and the development of peanut allergyTOULOUSE-INSA (315552106) / SudocSudocFranceF

Intact skin and not stripped skin is crucial for the safety and efficacy of peanut epicutaneous immunotherapy (EPIT) in mice

<p>Abstract</p> <p>Background</p> <p>Epicutaneous immunotherapy (EPIT) on intact skin with an epicutaneous delivery system has already been used in preclinical and clinical studies. In epicutaneous vaccination and immunotherapy, the stripping of skin before application of the allergen is suggested to facilitate the passage of allergen through immune cells.</p> <p>Objectives</p> <p>The aim of this study was to compare the immunological response induced by EPIT performed on intact and stripped skin in a mouse model of peanut allergy.</p> <p>Methods</p> <p>After oral sensitization with peanut and cholera toxin, BALB/c mice were epicutaneously treated using an epicutaneous delivery system (Viaskin® (DBV Technologies, Paris) applied either on intact skin or on stripped skin. Following EPIT, mice received an exclusive oral peanut regimen, aimed at triggering esophageal and jejunal lesions. We assessed eosinophil infiltration by histology, mRNA expression in the esophagus, antibody levels and peripheral T-cell response.</p> <p>Results</p> <p>EPIT on intact skin significantly reduced Th2 immunological response (IgE response and splenocyte secretion of Th2 cytokines) as well as esophageal eosinophilia (2.7 ± 0.9, compared to Sham 19.9 ± 1.5, p < 0.01), mRNA expression of Th2 cytokines in tissue and intestinal villus sub-atrophia (2.9 ± 0.2 vs Sham, 2.1 ± 0.2, p < 0.05). By contrast, EPIT on stripped skin reinforced Th2 systemic immunological response as well as eosinophil infiltration (26.8 ± 15.1), mRNA expression of Th2 cytokines and duodenal villus/crypt-ratio (2.4 ± 0.3).</p> <p>Conclusions</p> <p>Epicutaneous allergen-specific immunotherapy needs the integrity of superficial layers of the stratum corneum to warranty safety of treatment and to induce a tolerogenic profile of the immune response.</p

Treatment of colitis by epicutaneous immunotherapy in a murine model

Treatment of colitis by epicutaneous immunotherapy in a murine mode

Epicutaneous but Not Oral Immunotherapy Leads to Sustainable GATA-3 Hypermethylation and Foxp3 Hypomethylation in Peanut Sensitized Mice

International audienceRationaleEpicutaneous immunotherapy (EPIT) is a safe method for treating food allergies and animal models show that protection is sustainable. Previously, EPIT has been shown to alter epigenetic modifications and expression of Th2 and Tregs without influencing the expression of Th1 in peanut-sensitized mice. This study investigates the kinetics of epigenetic modifications underlying the therapeutic effect of EPIT and its persistence compared to oral immunotherapy (OIT).MethodsMice were orally sensitized to peanut and then treated by EPIT or OIT or non-treated (sham). Mice were sacrificed every 2 weeks during the immunotherapeutic protocols and also 8 weeks after the end of immunotherapy. DNA methylation was analysed in sorted CD4 T cells from spleen and blood by pyrosequencing.ResultsIn spleen and blood CD4 T-cells, significant hypermethylation of CpG islands of Gata3 was observed from the 4th week of EPIT and persisted following the end of treatment. This modification was not observed with OIT. In parallel, significant hypomethylation was observed in the Foxp3 CpG islands in spleen and blood CD4 T-cells from the 4th week of EPIT compared to Sham, which persisted following the end of treatment. For OIT, a similar level of hypomethylation was observed only in spleen CD4 T cells but was not sustained following the end of treatment.ConclusionsThe hypermethylation of Th2 transcription factor appears to be a specific trait of EPIT-induced immunomodulation. Foxp3 hypomethylation occurred with both EPIT and OIT, but proved sustainable only with EPIT, explaining the sustainability of EPIT protection in the mouse model

No impact of filaggrin deficiency on the efficacy of epicutaneous immunotherapy in a murine model

Background: Epicutaneous immunotherapy (EPIT&reg;), currently investigated in the treatment of food allergy, needs the integrity of the skin to warrant safety and efficacy. Mutations in the gene encoding the key epidermal protein filaggrin (FLG) are risk factors for peanut allergy and disrupt the skin intergrity. We investigated the association between FLG deficiency and peanut EPIT&reg; efficacy in a murine model. Methods: FLG mutant mice deficient in filaggrin (FLG-/-) or wild-type (WT) mice were sensitized with peanut protein extract (peanut protein) and cholera toxin. Sensitized mice received a patch per week during 8 weeks for EPIT&reg;, using Viaskin&reg;, and were then submitted to sustained peanut oral exposure. We assessed blood humoral and cellular responses and evaluated eosinophil infiltration in the gut mucosa. The different steps of allergen capture and transportation following deposition on the skin was also analyzed in sensitized mice. Results: Sensitization of mice was confirmed by a significant increase of specific Th2 biaised immunological responses. In sensitized mice, EPIT&reg; significantly reduced IgE levels, splenocytes secretion of Th2 cytokines and recruitment of eosinophils in esophagus, compared to sensitized mice without epicutaneous immunotherapy. The allergen applied onto the skin of FLG-/- mice did not undergo passive skin passage or systemic delivery. Instead, the allergen was captured by skin CD205high&nbsp;DCs, which migrated to afferent lymph nodes, as already described in WT mice. Conclusions: EPIT&reg; was efficient and safe in FLG-/- mice, suggesting that in Humans EPIT&reg; keeps efficacy and safety in the presence of loss of function of FLG