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Background: Epicutaneous immunotherapy (EPIT®), 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® 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®, using Viaskin®, 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® 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 CD205highDCs, which migrated to afferent lymph nodes, as already described in WT mice. Conclusions: EPIT® was efficient and safe in FLG-/- mice, suggesting that in Humans EPIT® keeps efficacy and safety in the presence of loss of function of FLG.

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.

Background 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. Objective To investigate the role of Tregs in EPIT and characterize their phenotype and maintenance following EPIT. Methods Tregs were investigated using in vivo depletion or adoptive transfer into BALB/c mice. Tregs were depleted using anti-CD25 antibody injection during EPIT, and allergen-specific responses were compared with Sham, EPIT alone and naive mice. To demonstrate that Tregs can mediate protection by their own, and to study their maintenance following the end of EPIT, CD25(+) CD4(+) Tregs isolated from mice just after or 8 weeks after EPIT were transferred into peanut-sensitized mice. Foxp3-IRES-mRFP mice were transferred with EPIT-induced Tregs to analyse the induction of host Tregs. Results The anti-CD25 antibody injection to EPIT mice abrogated the induction of Tregs in spleen and the expression of Foxp3 in oesophagus. This resulted in levels of peanut-induced eosinophilic infiltration in oesophagus similar to Sham and significantly higher than EPIT. Whereas the transfer of Tregs from Sham-treated mice demonstrated no effect, the transfer of Tregs isolated just after EPIT prevented peanut-induced eosinophil infiltration and eotaxin expression and induced Foxp3 in oesophagus. The transfer of Tregs isolated 8 weeks after EPIT suppressed allergen-specific responses as efficiently as did Tregs isolated just after EPIT and increased spleen Foxp3(+) CD25(+) CD4(+) cells similarly. The use of reporter mice demonstrated an increase in host Tregs. Conclusions These results confirm the Tregs-mediated mechanism of EPIT and demonstrate the persistence of efficient Tregs during a long period of time after treatment cessation. This suggests that EPIT induces long-term tolerance in peanut-sensitized mice.

Introduction L’immunotherapie epicutanee sur peau intacte (EPIT) est une voie therapeutique des allergies alimentaires en cours de developpement. Dans les modeles animaux, la desensibilisation obtenue par l’EPIT est maintenue apres arret du traitement (tolerance) et previent de sensibilisations a d’autres allergenes. Le but de ce travail etait d’evaluer, dans un modele de souris sensibilisees, la cinetique des modifications epigenetiques etayant l’effet therapeutique de l’EPIT et l’induction de tolerance. Methodes Des souris BALB/c femelles sensibilisees a l’arachide ont ete traitees par EPIT ou immunotherapie orale (OIT) ou non traitees pendant 8 semaines. Les prelevements de rate et sang ont ete realises aux semaines 1, 2, 4 et 8 du traitement et 8 semaines apres arret du traitement. Un autre panel de groupes de souris a ete analyse apres une phase de sensibilisation a un autre allergene, l’ovalbumine. La methylation de l’ADN a ete analysee par pyrosequencage dans les cellules CD4+ purifiees de la rate et du sang. Resultats Dans les cellules CD4+ (rate et sang), une hypermethylation des ilots CpG associes au promoteur de Gata3 apparait des la 4e semaine d’EPIT, persiste jusqu’a la fin du traitement (p Conclusion L’EPIT comparee a l’OIT conduit a une signature epigenetique unique et stable permettant une regulation negative de l’expression des genes de la voie du Th2 et une activation des cellules T regulatrices. Ces mecanismes seraient impliques dans l’induction de tolerance par EPIT.

Background Allergy to cow's milk increases the risk of sensitization to other foods in young children. Objectives We sought to evaluate the effect of early epicutaneous immunotherapy (EPIT) on further sensitization to peanut or house dust mite (HDM) in a murine model of sensitization to cow's milk. Methods BALB/c mice orally sensitized to milk were epicutaneously treated with a Viaskin patch (DBV Technologies) loaded with milk proteins for 8 weeks. Mice were then sensitized to peanut or HDM. After sensitization to peanut, mice were exposed to a peanut regimen known to induce eosinophilic esophageal inflammation. After sensitization to HDM, mice were challenged with aerosols to HDM, and airway hyperresponsiveness was evaluated by using plethysmography. Humoral response was also analyzed. The role of regulatory T (Treg) cells was evaluated by adoptively transferring Treg cells from milk EPIT–treated mice to naive mice before sensitization to peanut. Protection against anaphylaxis was also investigated. Methylation of the promoter region of transcription factors was analyzed by using PCR assays. Results In milk-sensitized mice specific EPIT prevented further sensitization to peanut or HDM. EPIT significantly modified the humoral response, reduced T H 2 cytokine levels, decreased eosinophilic esophageal infiltration, and suppressed airway hyperresponsiveness. The protective effect was sustained over 2 months. Moreover, the adoptive transfer of milk EPIT Treg cells completely prevented sensitization to peanut and peanut-induced anaphylaxis. Milk EPIT enhanced methylation of the GATA-3 promoter region. Conclusions Our results showed that EPIT influences the natural history of allergy and reduces the risk of further sensitization through a Treg cell–dependent mechanism.

Introduction L’immunotherapie epicutanee sur peau saine (EPIT®) constitue une voie therapeutique nouvelle des allergies alimentaires. Ses mecanismes d’actions ont ete mis en evidence dans plusieurs modeles precliniques et sont largement lies aux cellules T regulatrices (Tregs). Par son profil d’efficacite et sa securite d’emploi, elle est adaptee au traitement precoce des allergies. Le but de ce travail est d’evaluer, dans un modele de souris sensibilisees, l’impact du traitement precoce par EPIT sur l’evolution naturelle des allergies. Methodes Des souris BALB/c femelles sensibilisees au lait ont ete traitees par EPIT ou non traitees (NT) pendant 8 semaines avant d’etre sensibilisees a l’arachide. Les animaux ont ensuite ete testes par injection intraveineuse (IV) d’arachide afin de declencher une anaphylaxie evaluee par la baisse de temperature et la liberation de mMCP-1dans le plasma. Des souris uniquement sensibilisees a l’arachide et des souris naives constituent respectivement les groupes controles positifs et negatifs. Dans un deuxieme temps, des Tregs provenant de souris sensibilisees au lait EPIT ou NT ont ete transferees a des souris naives avant sensibilisation a l’arachide. La reponse a l’injection IV d’arachide a ete etudiee dans les memes conditions. Resultats Les souris NT sensibilisees successivement au lait puis a l’arachide presentaient, comme les souris sensibilisees a l’arachide, une forte chute de la temperature corporelle apres le challenge IV (4,1 °C ; p Conclusion Le traitement precoce par EPIT pourrait prevenir les sensibilisations ulterieures par un mecanisme dependant des Tregs. Des etudes cliniques devront confirmer cet effet protecteur chez l’Homme.

BackgroundFood 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.MethodsMice 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.ResultsSustained 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/mm(2)) 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/mm(2), pConclusionsGastro-intestinal lesions induced by sustained oral exposure in sensitized mice are efficaciously treated by allergen specific EPIT.

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

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.

Introduction Les cellules T regulatrices (Tregs) jouent un role primordial dans les mecanismes d’action de l’immunotherapie specifique. La capacite des Tregs a migrer vers les organes cibles est liee a l’expression de recepteurs specifiques. Le but de cette etude est la caracterisation de l’expression des recepteurs de migration vers la peau (CLA) et le tube digestif (CCR9) par les Tregs induit par l’immunotherapie epicutanee (EPIT) qui constitue une voie therapeutique nouvelle pour le traitement des allergies alimentaires. Methodes Des souris BALB/c femelles sensibilisees a l’arachide par voie orale ont ete traitees par EPIT ou non traitees (NT). Apres 8 semaines de traitement, les souris ont ete sacrifiees afin de prelever la rate, les ganglions mesenteriques (mLNs) et les ganglions inguinaux (iLNs) drainant la peau. Apres extractions des cellules, la proportion de Tregs et l’expression des recepteurs de migration specifique CLA + CCR9– (peau), CLA-CCR9+ (tube digestif) et CLA + CCR9+ (double tropisme) ont ete analyses par cytometrie en flux. Resultats Apres 8 semaines de traitement, la proportion de cellules Tregs CD4 + CD25 + Foxp3+ dans la rate et les iLNs est augmentee par EPIT par rapport au NT ( p p p p Discussion Les Tregs Foxp3+ jouent un role essentiel au cours du traitement de l’allergie alimentaire. Le tropisme a la fois digestif et cutane des Tregs induits par l’EPIT aussi bien dans les ganglions drainant la peau que le tube digestif, fait de cette methode un traitement de choix de l’allergie alimentaire.

Results: In spleen, expression of CCR4 increased on Tregs induced by all 3 therapies whereas the expression of CXCR3, CCR6 and CCR8 increased on EPIT-induced Tregs only. Skin homing receptor (CLA) increased on EPITbut not SLITor OIT-induced Tregs. Gut homing receptor CCR9 increased on EPITand OITbut not SLIT-induced Tregs. Interestingly, 81% of the CCR9+ Tregs induced by EPIT expressed CLA whereas only 43% did after OIT. In iLN, Tregs level increased only after EPIT with induction of CLA+CCR9-(22%) and CLA+CCR9+ (12%) Tregs. In mLN, EPIT and OIT significantly induced CCR9+ Tregs, 25% and 18% of them expressing also CLA in EPIT and OIT respectively.

Results: In splenocytes, a significant hypermethylation in GATA-3 CpG islands was induced by EPIT versus Sham, starting from the 4 of treatment (p

Methods: After sensitization to milk, mice were treated by EPIT or Sham, and 2 weeks later, submitted to a peanut-sensitization procedure. Mice were then intravenously challenged with peanut. In a second experiment, CD4+CD25+ T cells were isolated from spleen of milk-sensitized mice after EPIT or Sham, and transferred into naive mice. Recipient mice were submitted to peanut sensitization and intravenously challenged with peanut. In a third experiment, Tregs were obtained from Foxp3+-gfp mice sensitized to peanut and EPIT-treated, then adoptively transferred. Recipient mice were submitted or not to peanut sensitization before transfer, then all sensitized and IV challenged to ovalbumin. Outcome tools were rectal temperature, hypersensitivity reactions and blood mouse mast cell protease-1 (mMCP1).

Background 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. Objectives 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. Methods 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. Results 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 Conclusions 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.