Identification of a New Natural Ara h 6 Isoform and of Its Proteolytic Product as Major Allergens in Peanut

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MicroRNA-sequencing in a mouse model of peanut sensitized mice treated by EPIT identifies early changes in microRNA expression influencing T-cell plasticity and Th2 cytokine production

International audienceIntroduction:Epicutaneous immunotherapy (EPIT) is a safe treat-ment for food allergy. In animal models, EPIT protection seems toconfer sustained unresponsiveness and prevents further sensitiza-tion. We have previously shown DNA methylation changes in keytranscription factors to be associated with EPIT and sustained unre-sponsiveness.Objectives:This study investigates the kinetics of miRNA expres-sion patterns underlying the therapeutic effect of EPIT and its per-sistence. BALB/c mice were orally sensitized to peanut and thentreated with EPIT or not treated (sham). Mice (n=112) were sacri-ficed during treatment at 1, 2, 4, 6 and 8 weeks; and 8 weeks afterthe end of treatment. MiRNAs were analysed in sorted CD4+cellsfrom spleen using high-throughput sequencing on a HiSeq4000 to adepth of 200M bases / sample and validated by qPCR.Results:Global miRNA profiles consisting of 800-1000 miRNAsreproducibly detected in the CD4 samples distinguished EPIT-treatedmice from controls as early as one week following initiation of treat-ment. Intra-group variability of miRNA expression profiles was muchreduced in the EPIT group at all time points. Between 23 and 190MiRNAs were found to be differentially expressed at padj<.05 with alarge overlap of miRNAs between adjacent time points. Differentiallyexpressed miRNAs include miRNAs controlling T cell stability andplasticity (eg,Tregs, miR-10a) and Th2 cytokine production (eg, miR-92a-3p and miR-423-5p). 34 miRNAs were differentially expressedeight weeks after the end of the treatment, of which 24 (70%)showed similar expression changes at the end of treatment.Conclusions:EPIT leads to homogeneous changes in miRNAexpression profiles shortly after the initiation of treatment differenti-ating EPIT-treated mice from sham-treated control mice and expres-sion changes are maintained following the termination of treatment.Differentially expressed miRNAs include miRNAs in T cell plasticityand postulated targets include genes previously associated withallergy and asthma. Our study provides further evidence for themolecular alterations underlying sustained unresponsiveness in EPIT

Gata3

International audienceBackground 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