Discussion
In this study, we aimed to evaluate the potential of EPIT to treat cashew allergy. To that end, we first developed a clinically relevant mouse model of anaphylaxis, for which IgE is the main mediator and in which challenge is performed by oral administration of allergens, similar to what occurs in humans. Using this model, we demonstrated that epicutaneous patches are able to deliver cashew allergens to skin DCs, especially Langerhans cells and cDC2 that have been demonstrated as the main promotors of tolerance to topical allergens.14,15More importantly, EPIT with cashew patches affords a substantial level of protection against IgE-mediated anaphylaxis to cashew. In this study, we used a 50-µg dose per patch. This dose was selected based upon preliminary experiments, showing that 100, 250 or 500 µg doses did not afford additional benefits for protection (data not shown). This is, to our knowledge, the first study demonstrating the efficacy of a prototype immunotherapy against cashew allergy. Recently, Pereira et al. , evaluated the immunogenicity of orally-administered poly(anhydride) nanoparticles loaded with cashew allergens in BALB/c mice.16 These authors showed that nanoparticles were able to promote a strong specific Th1 response to cashew in association with an induction of FoxP3+ and LAP+ T regulatory cells (T-Reg). Unfortunately, the clinical efficacy of this treatment has not been reported. In our preclinical model, we showed that EPIT induced a transient peak of cashew-specific IgE at the beginning of immunotherapy. Interestingly, the same phenomenon has been observed in patients during clinical trials investigating EPIT against peanut allergy.17 Moreover, and in a similar way to what has been observed in humans, this increase is followed by a gradual progressive decrease of IgE titers with continued treatment. Additionally, our results revealed that EPIT induces an increase of cashew-specific IgG (IgG1, mouse equivalent of human IgG4 and IgG2a, mouse equivalent of human IgG1). Again, these data should be viewed in the context of clinical data investigating EPIT for peanut allergy, in which a progressive increase of peanut specific IgG4 levels has been observed during the first 12 months of treatment.17This increase in IgG4 response in humans is usually interpreted as a beneficial impact of the treatment since IgG4 antagonizes effector functions mediated by IgE through competition and neutralization of allergens.18 The permanent increase of IgG2a observed in our study should also be interpreted as a positive outcome since it is the main marker of Th1 immune orientation in mouse, which is known to be associated with non-allergic responses.19,20Mast-cells express a high amount of high affinity IgE receptors (FcεRI) and are unequivocally considered as key players in IgE-dependent anaphylaxis.21 Additionally, it has been previously shown that IgE-dependent activation of mast-cells plays an important role in disease induction in mouse models of allergy.22,23 Here, we demonstrated that EPIT strongly reduced mast-cell stimulation and degranulation following oral challenge. Interestingly, this inhibition occurs despite a relatively high level of IgE, suggesting that EPIT modulates mast-cell reactivity to IgE signaling. Beyond the competition between IgE and IgG for allergen binding we described above, this modulation could be linked to an inhibition of the signal triggered by FcεRI as described in previous studies.24,25 However, it is unlikely that this desensitization results from constant stimulation of gut mast-cell with small, sub-activating amounts of allergen, since EPIT does not lead to systemic diffusion of allergen. Therefore, we are currently performingin vivo experiments to elucidate the mechanisms underlying this modulation and the induction of tolerance to cashew, with a special focus on regulatory T-cells which have been demonstrated as key players in murine models of peanut allergy.26,27 Despite almost complete suppression of mMCP-1 and mMCP-7 levels in blood following 8 and 12 weeks of treatment, EPIT mice still presented substantial anaphylactic symptoms following oral challenge. Therefore, we cannot exclude a role for specific IgG in anaphylactic reactions, as suggested by Figure 2. Consequently, our mouse model may underestimate the potential efficacy of EPIT in humans, for which the role of IgG in anaphylaxis is still a matter of considerable debate.
EPIT may offer a safe approach to treat food allergy,28 primarily due to the absence of systemic dissemination of allergens, thus avoiding direct contact with anaphylaxis triggering effector cells, e.g. mast-cells. Therefore, we suggest that EPIT may be a relevant treatment strategy for cashew allergy. Indeed, the unique severity of reactions to cashew reported in allergic patients could be a source of concerns regarding other immunotherapeutic strategies that require subcutaneous injections or oral administration of allergens. A recent study demonstrated that many patients suffering from cashew allergy are IgE-sensitized to other nuts of the Anacardiaceae family.29 Thus, it would be interesting in future experiments to evaluate the capacity of cashew-specific EPIT to afford cross-protection against anaphylaxis to pistachio or mango, and therefore determine its spectrum of coverage among Anacardiaceae family members.
Overall, our data demonstrate that EPIT could be a relevant strategy to treat cashew allergy, bringing hope to patients suffering from this potentially life-threatening immune dysregulation.