The prevalence of allergic diseases, including food allergies, is increasing globally. Food allergies, characterized by IgE-mediated mast cell (MC) activation, can cause symptoms ranging from mild gastrointestinal distress to life-threatening anaphylaxis. Current treatments primarily focus on symptom management, such as inhibiting MC degranulation or blocking MC-derived mediators. Oral immunotherapy (OIT) is emerging as an effective approach to controlling food allergy by inducing a state of allergen unresponsiveness or tolerance. However, the detailed mechanisms of OIT, particularly the immunological transition from desensitization to long-term regulation, remain unclear. This necessitates further investigation to improve OIT efficacy and safety. Most studies have focused on peripheral blood analysis, neglecting the crucial role of the gut mucosa and its immune system. OIT comprises an initial escalation phase (acute desensitization) followed by a maintenance phase (consolidation). Successful desensitization of MCs is crucial for limiting allergic reactions and achieving long-term tolerance. Previous studies have hinted at immunomodulatory roles for MCs, producing regulatory cytokines. However, challenges in studying human mucosal tissue necessitate the use of animal models for mechanistic insights. This study aimed to establish a clinically relevant murine OIT model to investigate the functions of MCs in the transition from an allergic promoter to a suppressor state during OIT in the gut mucosa, focusing on the modulation of MCs and their interaction with regulatory T cells.

The literature extensively documents the increasing global prevalence of allergic diseases and the significant impact of food allergies. Studies highlight the role of IgE-mediated mast cell activation in the pathogenesis of food allergy, emphasizing the importance of MCs and their mediators in inducing gastrointestinal symptoms and potentially life-threatening anaphylaxis. Various symptomatic therapies target MC degranulation or mediator blockade. The role of MC-derived Th2 cytokines (IL-4, IL-5) in augmenting Th2 responses is also established. Allergen-specific immunotherapy, including subcutaneous, sublingual, and oral routes, has shown promise in reducing allergic reactions. While OIT is recognized as an effective approach, the underlying cellular and molecular mechanisms remain incompletely understood, particularly concerning long-term efficacy and safety. The precise immunological transition from the initial OIT phase to the maintenance phase, leading to unresponsiveness, needs further elucidation. Previous research has explored the novel immunomodulatory functions of MCs, including their production of regulatory cytokines. However, limitations in studying human mucosal tissues necessitate the use of animal models to unravel the intricate mechanisms governing OIT-mediated immune responses in the gut mucosa.

To investigate the role of mast cells in oral immunotherapy (OIT)-mediated control of food allergy, the researchers developed a murine model of OVA-induced allergic diarrhea. They utilized two MC-deficient mouse models (*KitW-sh/W-sh* and Mas-TRECK mice) to confirm the essential role of MCs in allergic diarrhea. An OIT protocol involving dose escalation of heated OVA was established and optimized to effectively inhibit allergic diarrhea. The researchers assessed the severity of allergic diarrhea based on stool consistency and water content, comparing OIT-treated mice with untreated allergic mice. The activation status of intestinal MCs in OIT mice was analyzed by measuring serum levels of mast cell protease-1 (mMCPT1), CD63 expression (a marker of degranulation), and the frequency of degranulated MCs. IL-4 expression in MCs was also examined by quantitative RT-PCR. To evaluate the role of regulatory T cells (Tregs) in OIT, the researchers examined the number of Tregs and Tr1 cells in OIT-treated mice and mice in which OVA administration had been stopped after desensitization. Anti-CD25 antibody was used to deplete Tregs in OIT-treated mice, assessing the impact on allergic diarrhea and OVA-specific IgE levels. To investigate the interaction between MCs and Tregs, the researchers examined the impact of MC depletion during OIT on Treg numbers and function. An in vitro desensitization protocol using IgE-bound bone marrow-derived MCs (BMMCs) was employed to analyze MC activation, cytokine production, and the effect of desensitized MCs on Treg expansion. Gene expression profiling and ChIP-Seq analysis of MCs from different conditions were performed to elucidate molecular mechanisms. The role of IL-2 in Treg induction by desensitized MCs was investigated using IL-2-neutralizing antibody. Finally, the researchers analyzed IL-2 and IL-10 expression in MCs isolated from the intestines of allergic and OIT mice. Statistical analyses were performed using unpaired, two-tailed Student's t-tests.

This study revealed that orally induced allergic diarrhea in mice required IgE-mediated mast cell (MC) activation. A dose-escalation oral immunotherapy (OIT) protocol effectively inhibited allergic diarrhea, correlating with a significant decrease in colonic MC numbers and reduced degranulation. Desensitized MCs showed decreased expression of CD63 and IL-4, indicating a reduction in activation and pathogenic Th2 responses. OIT induced significant expansion of Foxp3+ regulatory T cells (Tregs), while Tr1 cell numbers remained unchanged. Depletion of Tregs using anti-CD25 antibody reversed the protective effect of OIT, resulting in recurrence of allergic diarrhea and increased OVA-specific IgE production. Importantly, MC depletion during OIT drastically reduced Treg numbers and impaired Treg cytokine production (IL-10 and TGF-β), demonstrating the crucial role of MCs in Treg induction during OIT. In vitro desensitization of MCs resulted in reduced CD63 expression and β-hexosaminidase release, consistent with in vivo findings. Desensitized MCs, unlike allergic MCs, expanded Treg populations in vitro. This effect was mediated by secreted factors, particularly IL-2, as demonstrated by transwell assays and IL-2 neutralization experiments. Desensitized MCs exhibited increased IL-2 and IL-10 mRNA expression. These findings suggest a mechanism where OIT induces desensitized regulatory MCs which, in turn, promote Treg expansion through IL-2 production, leading to suppression of allergic responses.

This study provides compelling evidence for a novel regulatory network involving orally desensitized mast cells (MCs) and regulatory T cells (Tregs) in the control of food allergy. The findings highlight the crucial role of MCs not just as effectors of allergic reactions but also as potential mediators of tolerance. The established murine OIT model closely mimics the clinical scenario, enabling a detailed exploration of the immunological events within the gut mucosa. The observed decrease in MC degranulation and Th2 cytokine production during OIT, coupled with Treg expansion and the critical dependence of this expansion on MCs, strongly supports the idea that inducing regulatory MCs is a key mechanism of OIT-mediated protection. The in vitro studies confirmed the ability of desensitized MCs to directly promote Treg expansion, primarily through the secretion of IL-2. This IL-2-dependent pathway appears to be independent of IL-33, suggesting a specific mechanism for Treg induction in the context of OIT. These findings advance our understanding of the intricate interplay between MCs and Tregs in regulating food allergy and suggest novel therapeutic strategies targeting MC function.

This study demonstrates a critical role for orally desensitized mast cells in the successful outcome of oral immunotherapy for food allergy. The desensitized mast cells promote the expansion of regulatory T cells through IL-2 production, leading to suppression of allergic responses and long-lasting tolerance. This work highlights the potential of targeting mast cell function as a novel therapeutic strategy for treating allergic diseases.

The study utilized a murine model, which may not fully recapitulate the complexity of human food allergy. Further studies are needed to confirm these findings in human subjects. The specific molecular mechanisms underlying the conversion of mast cells from pathogenic to regulatory states require further investigation. The study focused primarily on gastrointestinal symptoms; further research is needed to explore the effects of this regulatory network on other manifestations of food allergy.