Efficacy of epicutaneous immunotherapy (EPIT) in a new model of peanut-induced eosinophilic esophagitis (EoE) and allergic enterpathy (AE)

Background Eosinophilia is often linked to allergic gastrointestinal disorders linked to food allergy. EPIT using Viaskin® device has been described as a therapeutic method in food allergy. We developed a model of mice sensitized to peanut, exhibiting EoE and AE after exclusive feeding with peanut protein extracts (PPE). This study was conducted in order to evaluate the efficacy of EPIT. Methods After oral sensitization with PPE and cholera toxin, 30 BALB/c mice were treated weekly during 8 weeks by PPE skin applications (EPIT), 20 mice were not treated (Sham) and 10 mice constituted the control group (C). Mice were then exclusively fed with PPE. Specific IgE, IgG1 and IgG2a were monitored during immunotherapy. Esophageal and jejunal samples were taken for histological analyses. Results sIgE increased after oral sensitization, respectively 0.207 ±0.03 and 0.214±0.04 μg/ml, in EPIT and Sham, with undetectable values in C. Following EPIT, sIgE decreased and sIgG2a increased, respectively 0.139±0.01 vs 0.166±0.01 μg/ml (EPIT vs Sham, p<0.05) and 14.96 ±0.60 vs 4.73±1.75 μg/ml (p<0.05). Esophageal eosinophilic infiltration (measured in 6 high power fields) was higher in Sham, 136±32, than in EPIT, 50±12 (p<0.05) and C, 7±3 cells/mm2 (p<0.01). Esophagus mucosa thickness was increased in Sham compared to EPIT and C (p<0.001). Sham group exhibited higher mRNA levels of cytokines than EPIT: eotaxin (p<0.05), IL-5 (p<0.05), IL-13 (p<0.05). The mRNA levels of these cytokines in EPIT were similar to C. The expression of Foxp3 mRNA increased significantky after EPIT compared with Sham and C (p<0.05). The jejunal villus/crypt ratio was lower in Sham than in EPIT and C, respectively 1.6 ±0.1 vs 2.3±0.2 (p<0.01) and 2.4±0.1 (p<0.001). Eosinophilic infiltration in jejunum was increased in Sham compared to EPIT (p<0.01) and C (p<0.001). Conclusion EPIT is effective in preventing EoE and AE induced by oral challenge in mice sensitized to peanut

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

Epigenetic Changes Following Epicutaneous Immunotherapy in Peanut Sensitized Mice

International audienceRationaleEpicutaneous immunotherapy (EPIT) is a promising route for treating food allergies and animal models show the sustainability of the protection. We investigated in peanut-sensitized mice the implementation of epigenetic mechanisms underlying this long-lasting therapeutic effect.MethodsMice were orally sensitized to peanut and then treated by EPIT or sham. Mice were sacrificed every 2 weeks during EPIT and also 8 weeks after the end of EPIT. DNA methylation was analysed in spleen and blood samples by restrictive enzyme digestion and quantitative-PCR and in sorted CD4, CD8 and CD19 cells from spleen and blood by pyrosequencing.ResultsIn splenocytes, a significant hypermethylation of the Gata3 CpG islands was induced by EPIT versus Sham, starting from the 4th of treatment (p<0.05). This hypermethylation was sustained after the end of EPIT. In circulating blood cells, the hypermethylation in the Gata3 CpG islands was observed only at the 8th week of EPIT (vs Sham, p<0.05). In spleen and blood CD4 cells, a significant hypermethylation for CpG island of Gata3 was observed from the 4th week of EPIT and persisted following the end of treatment. In parallel, a significant hypomethylation was obtained in the Foxp3 CpG island in spleen and blood CD4 cells from the 4thweek of EPIT compared to Sham, persisting after the end of treatment. No modification was observed for the Tbet transcription factor in whole or in sorted T and B cells sorted from spleen and blood.ConclusionsEpigenetic modifications of the DNA methylation of Th2 and Treg transcription factor appears a major trait of EPIT induced immunomodulation

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

Unique epigenetic modulation by EPIT compared to OIT in a model of peanut sensitized mice: sustainable GATA-3 hypermethylation and Foxp3 hypomethylation

International audienceBackground: Epicutaneous immunotherapy(EPIT) is a safe treatment for food allergy.In animal models, EPIT protection seemsto be more sustained compared to oralimmunotherapy (OIT) and prevents furthersensitization. This study investigates thekinetics of epigenetic modifications under-lying the therapeutic effect of EPIT, itspersistence and its bystander effect.Method: BALB/c mice were orally sensi-tized to peanut and then treated with EPITor OIT or non-treated. Mice were sacri-ficed during treatment, at 1, 2, 4, 6 and8 weeks; and 8 weeks after the end oftreatment. A set of peanut-sensitized micewere sacrificed following exposure to aprotocol of sensitization to a new allergen,OVA. DNA methylation was analysed insorted CD4+cells from spleen and bloodby pyrosequencing.Results: In spleen and blood CD4+cells,a significant hypermethylation of the CpGisland associated withgata3promoteroccurred at the 4th week of EPIT(P<0.05), persisted until the end of EPIT(P<0.05 andP<0.01, respectively) andwas sustained after the end of treatment(P<0.01). This change was not observedfor mice treated with OIT. A significanthypomethylation of theFoxp3CpG islandwas concomitantly observed in spleen andblood CD4+T cells, persisting until theend of EPIT (P<0.01) and sustained offtreatment(P<0.05).ForOIT,hypomethylation reached a level similarcompared to EPIT only in spleen CD4+Tcells and was not sustained off treatment. Interestingly, mice treated with EPIT andprotected from the sensitization to OVA asa new allergen maintained the epigeneticsignature characteristic for EPIT, i.e.Gata3hypermethylation (P<0.05) and Foxp3hypomethylation (0.05) in spleen and blood. No modification was observed forthe Tbet and RORg transcription factorswhatever the cells, organs or treatmentprotocol.Conclusion: EPIT, as compared to OIT,leads to a unique and stable epigenetic sig-nature with downregulation of Th2 DNAexpression and upregulation of Treg tran-scription factors, likely explaining the sus-tainability of the protection and itsbystander effec

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

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

Effect of EPIT on the induction of peanut-induced allergic esophagus inflammation established by histological and RT-qPCR analyses Microscopic analysis of eosinophils in the esophagus at 40× (A–C) and 100× high-powered fields (D–F).

<p>Most eosinophils (arrows) are located in the lamina propia, submucosa, and epithelial layer of the sham group and to a lesser extent of the EPIT group. A difference in the thickness of epithelium is observed between naive/EPIT and sham. Analysis of (G) chorion inflammation, (H) acanthosis, (I) measurement of eosinophil infiltration in the esophagus and in 40× high-powered fields. Grading for chorion inflammation is as follows: 0 = nothing, 1 = slight diffuse infiltration, 2 = perivascular area. Grading for acanthosis is as follows: 0 = nothing, 1 = layer with more than 4 cells, 2 = layer with more than 6 cells. The epithelium thickness was expressed as mean (µm) ±SD. For eosinophils, the results are expressed as number of eosinophils per mm² (means ± SD). Cytokine mRNA from esophagus segments collected 24 h after stopping peanut diet was assayed by RT-qPCR. Results are presented as mRNA expression of naive, Sham or EPIT animals. The relative levels of gene expression were calculated by reference to the mRNA levels of SDHA and β-actin in each sample. (J) eotaxin, (K) IL-5, (L) IL-13, (M) Foxp3, (N)GATA-3, (O) Tbet. * p<0.05, ** p<0.01.</p

Effect of EPIT on the induction of peanut-induced allergic enteropathy.

<p>(A–F) Jejunum segments collected and analyzed by microscopy after HES coloration (×40). Upper layer: villous atrophia could be observed in the sham group. Lower layer: inflammatory infiltration, particularly of eosinophils is shown. (G) Measurement of the ratio of villous height by crypt depth for each group of 10 mice under 10× high-powered fields. Results are expressed as means ± SD. ns: non significant, ** p<0.01, *** p<0.001.</p