Vincent Dioszeghy

Epicutaneous immunotherapy on intact skin using a new delivery system in a murine model of allergy

Background Allergen‐specific immunotherapy, subcutaneous immunotherapy (SCIT) or oral, has been used for almost a century to redirect inappropriate immune responses in atopic patients. A new mode of administration through the intact skin [epicutaneous immunotherapy (EPIT)], using an original epicutaneous delivery system, may represent an alternative to these classical methods.

Epicutaneous Immunotherapy Results in Rapid Allergen Uptake by Dendritic Cells through Intact Skin and Downregulates the Allergen-Specific Response in Sensitized Mice

Epicutaneous immunotherapy onto intact skin has proved to be an efficient and safe alternative treatment of allergy in an animal model with various allergens and in children for cow’s milk allergy. The aim of this study was to analyze the different steps of the immunological handling of the allergen when deposited on intact skin using an epicutaneous delivery system and its immune consequences in sensitized BALB/c mice. As expected, when applied on intact skin, OVA exhibits neither a passive passage through the skin nor any detectable systemic delivery. The current study demonstrates that, after a prolonged application on intact skin, OVA is taken up by dendritic cells in the superficial layers of the stratum corneum and transported, after internalization, to the draining lymph nodes, with variations according to the previous level of sensitization of the mice. When OVA is applied with the epicutaneous delivery system repeatedly, specific local and systemic responses are down-modulated in association with the induction of regulatory T cells. Besides providing new insights into skin function in the presence of allergens, this study indicates that the skin might have a tolerogenic role, at least when kept intact.

Epicutaneous Immunotherapy Using a New Epicutaneous Delivery System in Mice Sensitized to Peanuts

Background: Peanut allergy is a life-threatening condition for which new efficient and safe treatment is expected. We evaluated epicutaneous immunotherapy (EPIT) as a new alternative treatment for peanut allergy in sensitized mice. Methods: Sixty BALB/c mice were sensitized by gavages with peanut protein extract (PPE) mixed with cholera toxin. An epicutaneous delivery system, coated with 100 µg PPE (Viaskin®, DBV Technologies, Paris, France), was applied to intact skin every week during 48 h (EPIT; n = 20). This group was compared with sensitized mice treated with subcutaneous immunotherapy (SCIT; n = 20), untreated sensitized mice (sham, n = 20), and naive mice (naive; n = 20). After the 8-week treatment, a histamine release test, airway hyperreactivity measurement by plethysmography, and a resistance-compliance measurement after the challenge were performed. Blood and bronchoalveolar lavage were sampled for serology, cytokines, and cytology. Results: Specific IgE (sIgE) increased after sensitization in the EPIT (0.26 µg/ml) and SCIT (0.21 µg/ml) groups and decreased after treatment (0.09 µg/ml, p < 0.001 and 0.06 µg/ml, p < 0.001, respectively). The IgG1/IgG2a ratio decreased in the EPIT and SCIT groups versus the sham group (3.7; p < 0.001 and 2.7; p < 0.01 and 15.1, respectively). At the higher metacholine concentration, enhanced pause values were lower in the EPIT and SCIT groups than in the sham group (7.29, 6.74, and 10.99, p < 0.01, respectively), and did not differ from that of the naive group (5.06). Resistance-compliance was reversed in the treated groups versus the sham group (p < 0.001). IL-4, IL-5, IL-13, eotaxin, and eosinophils were reduced in the BAL of the EPIT and SCIT groups versus the sham group (p < 0.001). Conclusion: In peanut-sensitized mice, based on biological and physiological responses, EPIT is as efficient as subcutaneous treatment which is the reference method in immunotherapy.

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 (1.6±0.15). 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 (2.3±0.15). 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.

The regulatory T cells induction by epicutaneous immunotherapy is sustained and mediates long‐term protection from eosinophilic disorders in peanut‐sensitized mice

Allergen‐specific immunotherapy favours immune deviation from a Th2 to a Th1 response and increases the number of regulatory T cells (Tregs). Epicutaneous immunotherapy (EPIT) of sensitized mice decreases the clinical and the allergen‐specific Th2 responses and increases local and peripheral Foxp3+ Tregs.

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

BackgroundEpicutaneous 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.ObjectivesThe 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.MethodsAfter 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.ResultsEPIT 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).ConclusionsEpicutaneous 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.

Differences in phenotype, homing properties and suppressive activities of regulatory T cells induced by epicutaneous, oral or sublingual immunotherapy in mice sensitized to peanut.

Allergen-specific immunotherapy has been proposed as an attractive strategy to actively treat food allergy using the following three different immunotherapy routes: oral (OIT), sublingual (SLIT) and epicutaneous (EPIT) immunotherapy. Regulatory T cells (Tregs) have been shown to have a pivotal role in the mechanisms of immunotherapy. The aim of this study was to compare the phenotype and function of Tregs induced in peanut-sensitized BALB/c mice using these three routes of treatment. We show that although EPIT, OIT and SLIT were all able to effectively desensitize peanut-sensitized mice, they induced different subsets of Tregs. Foxp3+ Tregs were induced by the three treatment routes but with greater numbers induced by EPIT. EPIT and OIT also increased the level of LAP+ Tregs, whereas SLIT induced IL-10+ cells. The suppressive activity of EPIT-induced Tregs did not depend on IL-10 but required CTLA-4, whereas OIT acted through both mechanisms and SLIT was strictly dependent on IL-10. Moreover, the three routes influenced the homing properties of induced Tregs differently, with a larger repertoire of chemokine receptors expressed by EPIT-induced Tregs compared with OIT- and SLIT- induced cells, resulting in different protective consequences against allergen exposure. Furthermore, whereas OIT- or SLIT-induced Tregs lost their suppressive activities after treatment was discontinued, the suppressive activities of EPIT-induced Tregs were still effective 8 weeks after the end of treatment, suggesting the induction of a more long-lasting tolerance. In summary, EPIT, OIT and SLIT mediated desensitization through the induction of different subsets of Tregs, leading to important differences in the subsequent protection against allergen exposure and the possible induction of tolerance.

Epicutaneous Immunotherapy Compared with Sublingual Immunotherapy in Mice Sensitized to Pollen (Phleum pratense)

Background. The aim of this study was to compare the efficacy of epicutaneous immunotherapy (EPIT) to sublingual immunotherapy (SLIT) in a model of mice sensitized to Phleum pratense pollen. Methods. BALB/c mice were sensitized by sub-cutaneous route to pollen protein extract mixed treated for 8 weeks, using sham, EPIT, or SLIT. Measurements involved the serological response and cytokine profile from reactivated splenocytes, plethysmography after aerosol challenge to pollen, cell, and cytokine contents in the bronchoalveolar lavages (BALs). Results. After immunotherapy, sIgE was significantly decreased in the treated groups compared to sham (P < 0.001), whereas sIgG2a increased with EPIT and SLIT (P < 0.001 and P < 0.005 versus sham). Reactivated splenocytes secreted higher levels of Th2 cytokines with sham (P < 0.01). Penh values were higher in sham than EPIT and SLIT. Eosinophil recruitment in BAL was significantly reduced only by EPIT (P < 0.01). Conclusion. In this model of mice sensitized to pollen, EPIT was at least as efficient as SLIT.

Epicutaneous immunotherapy for food allergy as a novel pathway for oral tolerance induction

Epicutaneous immunotherapy is a developing technique, aiming at desensitizing patients with food allergy with less risks that oral ingestion or injection could generate. Several clinical trials have been performed and are currently running, in milk and peanut allergy, assessing the safety of the technique and its efficacy. Preclinical models indicate a major role in the mechanisms of desensitization, for example, Tregs and epigenetic modifications.

Gata3 hypermethylation and Foxp3 hypomethylation are associated with sustained protection and bystander effect following epicutaneous immunotherapy in peanut-sensitized mice

Epicutaneous immunotherapy (EPIT) is a promising method for treating food allergies. In animal models, EPIT induces sustained unresponsiveness and prevents further sensitization mediated by Tregs. Here, we elucidate the mechanisms underlying the therapeutic effect of EPIT, by characterizing the kinetics of DNA methylation changes in sorted cells from spleen and blood and by evaluating its persistence and bystander effect compared to oral immunotherapy (OIT).