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

BACKGROUND: 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). METHODS: BALB/c mice orally sensitized to peanut proteins (PPE) were treated by EPIT using a PPE-patch or by PPE-OIT. Another set of peanut-sensitized mice treated by EPIT or OIT were sacrificed following a protocol of sensitization to OVA. DNA methylation was analyzed during immunotherapy and 8 weeks after the end of treatment in sorted cells from spleen and blood by pyrosequencing. Humoral and cellular responses were measured during and after immunotherapy. RESULTS: Analyses showed a significant hypermethylation of the Gata3 promoter detectable only in Th2 cells for EPIT from the 4th week and a significant hypomethylation of the Foxp3 promoter in CD62L+ Tregs, which was sustained only for EPIT. In addition, mice treated with EPIT were protected from subsequent sensitization and maintained the epigenetic signature characteristic for EPIT. CONCLUSIONS: Our study demonstrates that EPIT leads to a unique and stable epigenetic signature in specific T-cell compartments with downregulation of Th2 key regulators and upregulation of Treg transcription factors, likely explaining the sustainability of protection and the observed bystander effect.

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

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 naïve 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.

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. OBJECTIVE: Proof of concept of efficacy of EPIT on intact skin in mice sensitized to aeroallergens or food allergens. METHODS: Mice were sensitized to pollen (n=18), house dust mite (HDM, n=24), ovalbumin (OVA, n=18) or peanut (n=18), and allocated to four groups: EPIT, SCIT, not treated (NT) and control. Specific Ig (sIg)E, sIgG1 and sIgG2a were monitored. After 8 weeks of treatment, plethysmography was performed after aerosol provocation with appropriate allergens. RESULTS: At the highest doses of methacholine, pause enhancement (Penh) values were significantly decreased in the EPIT group vs. the sensitized NT groups (7.5 vs. 12.3 - pollen, 7.6 vs. 8.9 - HDM, 11.5 vs. 14.5 - OVA, 7.6 vs. 12.8 - peanut, respectively) (P<0.05). With all the allergens tested, Penh values were similar in SCIT, EPIT and control. IgG2a for pollen, HDM, OVA and peanuts were significantly increased in the EPIT group vs. NT: 0.97 vs. 0.42 microg/mL, 2.5 vs. 0.46 microg/mL, 0.39 vs. 0.05 microg/mL and 15.0 vs. 5.5 microg/mL, respectively (P<0.05). There were no significant differences between EPIT and SCIT groups. The IgE/IgG2a ratio decreased significantly in the EPIT group for the four allergens from 70 to 58 (pollen), 175 to 26 (HDM), 5433 to 120 (OVA) and 49 to 6 (peanut), respectively (P<0.05). CONCLUSION: In mice sensitized to the four allergens tested, EPIT was as efficacious as SCIT, considered as the reference immunotherapy. These first results have to be confirmed by clinical studies.