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

Abstract

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 < 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).

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.

Figure 1

a-Study design for the evaluation of peanut protein passage into blood stream after epicutaneous application on intact or stripped skin. Naive mice were divided into 3 groups (n=10 for each). One group received a Viaskin® loaded with 500μg (Viaskin®-500) applied on intact skin (EPIT), another group received Viaskin®-500 applied on stripped skin (stripping+EPIT) and the last one received a subcutaneous injection containing 500μg of PPE. Blood samples were taken at different time points (0, 2, 8, 24, 48h) to quantify Ara h 1 in serum. b- Study design for the sensitization of mice to peanuts proteins and evaluation of the effect by EPIT on intact or stripped skin on the induction of digestive lesions on esophagus and jejunum. Twenty-four mice were sensitized to peanut proteins. Then, epicutaneous immunotherapy was conducted for 8 weeks in 8 sensitized mice on intact skin (EPIT), or in 8 sensitized mice on stripped skin (stripping+EPIT) and 8 other sensitized mice received a Sham treatment (Sham). After an oral challenge with high amounts of peanut proteins, histamine release was measured in blood samples. After that, a peanut regimen for 10 days was given to sensitized and naive mice. Mice were sacrificed to analyze esophagus and jejunum samples by histology and RT-qPCR. Blood samples were taken before the beginning of immunotherapy and after the 8-week period of treatment to measure specific immunoglobulins (IgE, IgG1, IgG2a).

Figure 2

Quantification of Ara h 1 in serum sample of mice. Quantity of Ara h 1 was measured in serum samples after epicutaneous administration on intact or stripped skin or subcutaneous administration of 500μg of PPE. Results were expressed in ng/ml as means ± SD for each group.

Figure 3

Systemic responses induced in mice after oral sensitization and epicutaneous immunotherapy (a) Quantity of specific IgE and (b) specific IgG2a expressed in μg/ml. Data are expressed as means ± SD for each group, D42 after oral sensitization, D106 after immunotherapy and sustained peanut exposure. (c) Measurement of histamine release in bloodstream after oral challenge to peanuts. Data are expressed as means ± SD for each group. * p<0.05, ** p<0.01, *** p<0.001.

Figure 4

Cellular responses induced in mice after oral sensitization and epicutaneous immunotherapy. Measurement of Th2 cytokine levels (IL-4, IL-5, IL-13) and IFN-γ secretion by splenocytes collected from each group of mice (naive, Sham, EPIT, stripping-EPIT) immediately after sacrifice. Splenocytes were prepared and stimulated with PPE for 72 hrs. Cytokines were measured by Bioplex cytokine assay®. Data are presented as means ± SD for each group, * p<0.05, ** p<0.01.

Figure 5

Effect of EPIT on intact or stripped skin on the induction of injuries in esophagus. Microscopic analysis of eosinophils in the esophagus at 100x high-powered fields (a-d). Most eosinophils are located in the lamina propria, submucosa and epithelial layer of the Sham and stripping-EPIT groups and to a lesser extend of the EPIT group. A difference in the thickness of epithelium is observed between naïve/EPIT and Sham/stripping+EPIT. (e) For eosinophils, the results are expressed as number of eosinophils per mm² and data are presented as means ± SD for each group, * p<0.05, ** p<0.01.

Figure 6

Effect of EPIT on intact or stripped skin on the mRNA expression of cytokines and transcription factors in esophagus mucosa. Cytokine mRNA from esophagus segments collected 24 hrs after stopping peanut diet was assayed by RT-qPCR. Results are presented as mRNA expression of naïve, Sham, or EPIT animals. The relative levels of gene expression were calculated by reference to mRNA levels of SDHA and β-actin in each sample. (a) eotaxin, (b) IL-5, (c) IL-13, (d) T-bet, (e) GATA-3, (f) FoxP3. * p<0.05, ** p<0.01 , *** p<0.001.

Figure 7

Effect of EPIT on intact or stripped skin on the induction of jejunal lesions jejunum segments collected and analyzed by microscopy after HES coloration (x40). (a-d) Inflammatory infiltration, particularly of eosinophils is shown. (e) quantification of the eosinophilic infiltration into jejunal mucosa. The results are expressed as number of eosinophils per mm² and data are presented as means ± SD for each group, * p<0.05, *** p<0.001.

Figure 8

Effect of EPIT on intact or stripped skin on the induction of villus sub-atrophia. Measurement of the ratio of the villous height by crypt depth for each group under 10x high powered fields. Results are expressed as means ± SD, * p<0.05, *** p<0.001.