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. Author manuscript; available in PMC: 2015 Jul 17.
Published in final edited form as: Immunity. 2014 Jul 10;41(1):141–151. doi: 10.1016/j.immuni.2014.05.017

Inhibition of Immunoglobulin E signals during allergen ingestion leads to reversal of established food allergy and induction of regulatory T cells

Oliver T Burton 1,2,†, Magali Noval-Rivas 1,2,†, Joseph S Zhou 1,2, Stephanie L Logsdon 1,2, Alanna R Darling 1, Kyle J Koleoglou 1, Axel Roers 3, Hani Houshyar 4, Michael A Crackower 4,5, Talal A Chatila 1,2, Hans C Oettgen 1,2
PMCID: PMC4123130  NIHMSID: NIHMS608517  PMID: 25017467

SUMMARY

Immunoglobulin E (IgE) antibodies are known for triggering immediate hypersensitivity reactions such as food anaphylaxis. In this study, we tested whether they might additionally function to amplify nascent antibody and T helper 2 (Th2) cell-mediated responses to ingested proteins and if blocking IgE would modify sensitization. Using mice harboring a disinhibited form of the IL-4 receptor, we developed an adjuvant-free model of peanut allergy. Mast cells and IgE were required for induction of antibody and Th2 cell-mediated responses to peanut ingestion and they impaired regulatory T (Treg) cell induction. Mast cell-targeted genetic deletion of the FcεRI signaling kinase, Syk, or Syk blockade also prevented peanut sensitization. In mice with established allergy, Syk blockade facilitated desensitization and induction of Treg cells, which suppressed allergy when transferred to naïve recipients. Our study suggests a key role for IgE in driving Th2 cell and IgE responses while suppressing Treg cells in food allergy.

INTRODUCTION

Food allergy has emerged as a major public health issue worldwide (Burks et al., 2012). Sensitized individuals, who have high titers of food-specific IgE antibodies, experience a range of immediate hypersensitivity responses including acute onset itching, hives, vomiting and diarrhea all triggered when food proteins recognized by FcεRI-bound IgE induce mast cell activation. The most dramatic food hypersensitivity reaction is systemic anaphylaxis, in which vasoactive mast cell mediators induce plasma extravasation, shock, cardiopulmonary collapse and death (Finkelman, 2007; Simons, 2010). The standard of care, namely recommendation to strictly avoid foods to which they are allergic, paradoxically deprives patients of the chance to naturally develop oral tolerance, as would likely occur if they were able to continue to ingest them without experiencing harmful effects.

Following the very successful example of subcutaneous immunotherapy in which subjects with aeroallergen sensitivity are desensitized by injection of protein extracts (“allergy shots”), investigators have evaluated oral desensitization strategies for food allergy (Nowak-Wegrzyn and Sampson, 2011; Vickery and Burks, 2010). Although achieving substantial success, such approaches are associated with unpredictable IgE antibody-mediated allergic reactions limiting their application in practice. Several groups have now performed oral desensitization under cover of the monoclonal anti-IgE antibody, omalizumab, which effectively blocks food anaphylaxis, with the expectation that inhibiting IgE-mediated reactions would improve the patient experience (Nadeau et al., 2011; Schneider et al., 2013).

A growing body of evidence indicates that IgE antibodies and mast cells might serve not only as the effectors of immediate hypersensitivity in subjects with established sensitivity but also as amplifiers during initial antigen exposure in naïve subjects, potentially providing early signals for nascent Th2 cell and antibody responses. IgE induces mast cell production of both Th2 cell-inducing and DC-activating cytokines (Asai et al., 2001; Kalesnikoff et al., 2001; Kawakami and Kitaura, 2005). We have reported that IgE and mast cells enhance immune sensitization in contact sensitivity (Bryce et al., 2004). Using an adjuvant-free asthma model, Galli and colleagues demonstrated that the induction of airway inflammation and bronchial hyper-reactivity are strongly amplified by mast cells in the airway mucosa (Nakae et al., 2007a; Nakae et al., 2007b). Additional evidence that effector cells of allergic responses might independently function as inducers of immune sensitization comes from studies implicating basophils (which, like mast cells, are FcεRI+) as early drivers of Th2 cell expansion. (Mukai et al., 2005; Sokol et al., 2009). We similarly hypothesized that IgE antibodies and mast cells might serve not only as the effectors of food anaphylaxis but also as important early inducers of Th2 cell responses and suppressors of Treg cell responses to food allergens and that they might provide an accessible therapeutic handle by which to dampen such responses.

Evaluation of this hypothesis required an animal model of food allergy in which immune sensitization could be accomplished directly by enteral sensitization and in the absence of the immunostimulatory effects of prior systemic parenteral immunization or ingestion of immunological adjuvants commonly used in food allergy models. For this purpose we took advantage of inherently atopic mice in which a mutation (F709) of the IL-4 receptor α-chain (IL-4rα) ITIM results in amplified signaling upon interaction with IL-4 or IL-13, but not constitutive activation. These mice exhibit enhanced Th2 cell responses and IgE production, and susceptibility to anaphylaxis following ingestion of the model antigen ovalbumin (OVA) in the absence of adjuvant (Mathias et al., 2011). We have now adapted this model to the clinically relevant food allergen, peanut, and have applied multiple parallel approaches that together provide strong evidence that IgE antibodies and mast cells enhance Th2 cell responses to ingested allergens. The mechanism for this Th2 cell induction is the suppression of Foxp3+ Treg cell responses, both in terms of Treg cell numbers and function. We show that established allergic sensitivity can be reversed when oral immunotherapy is paired with interventions that block IgE-mediated mast cell activation and demonstrate that pharmacologic inhibition of Syk activity can be used to inhibit mast cell function and achieve the same benefit as IgE blockade. Our results establish that IgE antibodies, signaling via FcεRI on mast cells, not only serve to induce anaphylactic reactions in individuals with food allergy but also exert critical regulatory effects on adaptive immunity to ingested proteins in emerging food allergy. Mast cells activated by IgE support Th2 cell responses and IgE production while suppressing Treg cell induction. Moreover, food ingestion under cover of IgE signaling blockade can lead to reversal of Th2 cell responses and induction of Treg cells in mice with established food allergy. These results suggest that pharmacologic IgE blockade might be of benefit to patients with food allergy.

RESULTS

IgE antibodies amplify Th2 cell responses and suppress Treg cell induction to ingested peanut

Whereas IgE antibodies are best recognized for their unique ability to trigger immediate hypersensitivity reactions in previously sensitized subjects there is evidence that they might serve an independent and important function in amplifying and skewing developing adaptive immune responses following initial antigen exposure (Gould and Sutton, 2008). For this study, we examined the possibility that IgE antibodies, signaling via FcεRI on mast cells, would support Th2 cell responses to ingested food allergens while suppressing the induction of Treg cells. In order to test this hypothesis, we developed a murine model of allergy to peanut, a very common food allergen and one associated with particularly severe anaphylactic reactions. We have previously reported the construction of mice (Il4ratm3.1Tch), which harbor a targeted insertion of a mutant form of IL-4rα in which the immunoreceptor tyrosine-based inhibition motif (ITIM) motif is disrupted (F709 mutation). These mice have normal IL-4rα expression and do not have baseline signaling but exhibit enhanced signaling on ligand exposure and are highly susceptible to the induction of allergy to ovalbumin (OVA) in an adjuvant-free ingestion model (Mathias et al., 2011; Tachdjian et al., 2010). They exhibit robust anaphylaxis following food allergen ingestion, a cardinal phenotype of food allergy that has previously been difficult to reproduce in animal models. We reasoned that these mice would provide an ideal tool for analysis of IgE and mast cell roles in food allergen sensitization to a physiologically relevant allergen, peanut.

Mice were sensitized by weekly gavage with 23 mg commercial peanut butter (5 mg protein) (Figure S1). After four doses, peanut-fed mice carrying the IL-4rα F709 mutation but not control animals exhibited strong peanut-specific IgE and IgG1 responses as well as robust induction of peanut-specific Th2 cell responses (Figure 1A, B, C, D and Figure S2A). Consistent with the expected induction of oral tolerance, wild-type BALB/c mice developed a strong peanut-specific Foxp3+ Treg cell response (Figure 1E). Peanut-specific Treg cell induction was markedly impaired in mice with the F709 IL-4 receptor. In this study we measured Treg cell responses by ex vivo stimulation for 5 days with peanut antigen followed by flow cytometric evaluation of Foxp3+ dividing cells.

Figure 1. Effects of IgE and IL4ra genotypes on food allergen sensitization in the absence of adjuvant.

Figure 1

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The effects of IgE on sensitization were evaluated using completely IgE-deficient mice (Igh-7−/−) both on the allergy-prone IL-4rα F709 background (Il4ratm3.1Tch) and on wild-type BALB/c. A) Peanut-specific IgE levels in sera of mice subjected to enteral gavage with peanut butter (PN). Mice (n=5–10 per group) were gavaged with 23mg PN once a week for four weeks. The horizontal line indicates the limit of detection in this ELISA. Presence of the activating IL-4 receptor, F709, and IgE are indicated by “+” or “−”. B) Serum titers of peanut-specific IgG1 after sensitization. C) Flow cytometric analysis of intracellular IL-4 staining in CD4+ T cells from mesenteric lymph node (MLN) cells after PN sensitization. D) IL-4 levels as determined by ELISA in splenocyte cultures from PN-treated mice re-stimulated in vitro with PN extract for five days. E) Analysis of PN-specific Treg cells expanded from MLN cells of PN-treated mice. F) Representative flow cytometry plots demonstrate dye dilution (proliferation) among CD3ε+CD4+ MLN cells labeled with Violet CellTrace and cultured in vitro with PN extract for five days. The summary graph shows the Foxp3 phenotype of the divided cells, gating on CD4+ viable single cells. G) Temperature curves following enteral challenge with high-dose PN. P<0.0001 by repeated measures (RM) two-way ANOVA, Il4ratm3.1Tch vs. Il4ratm3.1Tch Igh-7−/−. H) Serum MMCP-1 levels as measured by ELISA post-challenge. Data are represented as points for individual mice, with mean ±SEM in overlay. Similar data were obtained in at least three separate experiments.

Gavage challenge of peanut-sensitized mice with 450 mg peanut butter (100mg protein) elicited anaphylaxis in the IL-4rα mutants, measured using implanted thermal transponders, as loss of core body temperature (Figure 1G, see Figure S1 for protocol schematic). Neither unsensitized IL-4rα mutant mice nor peanut-fed wild-type BALB/c mice had any temperature loss (Figure 1G and data not shown), and challenge of peanut-sensitized mutant mice with irrelevant protein had no effect on temperature (Figure S2B). Anaphylaxis was accompanied by elevated serum concentrations of mouse mast cell protease (MMCP)-1, a marker of mucosal mast cell activation (Figure 1H). These findings demonstrate that mice with dysregulated F709 IL-4 receptor develop intense Th2 cell, IgE and IgG responses to ingested peanut along with a defective induction of Treg cells and that this immunological profile renders them exquisitely sensitive to anaphylaxis following enteral peanut challenge. It is interesting to note that in addition to being susceptible to deliberate induction of sensitization with the food allergen peanut, these mice exhibit some spontaneous sensitization to food proteins within their standard chow diet (Figure S2C).

In order to evaluate the contributions of IgE antibodies to peanut sensitization and anaphylaxis we crossed the IL-4rα mutant animals with IgE-deficient (Igh7−/−) BALB/c mice (Oettgen et al., 1994). As expected, IgE-deficient mice were fully resistant to peanut anaphylaxis (Figure 1G). Consistent with our hypothesis that IgE contributes to immunological sensitization in the gut during recurrent allergen ingestion, peanut specific IgG1 and Th2 cell responses were dramatically reduced in peanut-sensitized mice lacking IgE (Figure 1B, C, D). Treg cell induction, which had been markedly impaired in mice with the F709 form of IL-4rα, was fully restored in the absence of IgE (Figure 1E, F). Taken together, these findings provide strong evidence that IgE antibodies support the induction of Th2 cell and antibody responses to ingested allergens while suppressing the induction of Treg cells.

Our results with IgE-deficient mice suggest that the application of IgE blockade, which has been employed in clinical food desensitization studies to block undesired anaphylactic reactions, might also modulate immune responses to ingested antigens. Omalizumab, the anti-IgE used in human trials, is “nonanaphylactogenic.” It does not bind to FcεRI-bound IgE and hence does not activate mast cells. In contrast, available anti-mouse IgE reagents all exhibit some degree of mast cell activation. In order to use these antibodies in the peanut model we had to mitigate this effect. Taking advantage of a rush desensitization protocol developed by Khodoun and colleagues (Khodoun et al., 2013), we treated mice with anti-IgE in a four-day build-up prior to the first exposure to peanut. Neutralization of IgE mimicked genetic deletion of IgE, rendering the IL-4rα mutant mice resistant to anaphylactic reactions (Figure 2A). Anti-IgE treatment prior to sensitization reduced Th2 cell responses and increased peanut-specific Treg cell frequencies (Figure 2B, D, Figure S2D).

Figure 2. Effects of anti-IgE on food allergen sensitization.

Figure 2

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A) Temperature curves following acute enteral challenge with PN in IL-4rα F709 mutant (Il4ratm3.1Tch) mice (n=5) treated with PN and/or anti-IgE. Mice were sensitized by enteral gavage with PN (23mg) once a week for four weeks, followed by desensitization with PN (225mg) once a day for three weeks. Anti-IgE (100μg total) was injected i.p. in an incremental rush protocol prior to sensitization or desensitization. PN+αIgE: αIgE prior to PN sensitization; PN, PN+αIgE: PN sensitization, αIgE prior to PN desensitization; PN, PN: PN sensitization, PN desensitization. PN vs. PN+αIgE: P=0.0011; PN vs. PN, PN+αIgE: P=0.0022; PN, PN vs. PN, PN+αIgE: P=0.0002 by RM two-way ANOVA. B) Flow cytometric analysis of IL-4 by intracellular staining of CD4+ T cells in MLN (n=4–5). C) IL-4 staining in CD4+ MLN cells in response to desensitization with PN ±anti-IgE (n=5–7). D) Flow cytometric analysis of Foxp3+ cells in the CD3ε+CD4+ MLN cells exhibiting proliferative responses to PN after in vitro re-stimulation with 100μg/ml PN for 5 days (n=4–9). Data are from one of two experiments, and are represented as mean ±SEM.

Anti-IgE therapy in the peanut allergy model allowed us to also examine the effects of IgE blockade during allergen desensitization in animals with established peanut sensitivity. For desensitization, anti-IgE treatment was started a week after the final sensitizing peanut dose was administered, after which large doses (225 mg) of peanut were administered daily for three weeks. When finally challenged with peanut, mice that had received anti-IgE did not anaphylax, as expected given the requirement for IgE in anaphylaxis (Figure 2A). In contrast, mice subjected to the same peanut desensitization therapy without anti-IgE exhibited similar anaphylactic reactions to those seen in mice that received no therapy whatsoever. Th2 cell responses were reduced in mice treated with anti-IgE plus peanut, and peanut-specific Treg cell frequencies markedly increased (Figure 2C, D, Figure S2E). Control desensitization therapy with peanut alone did not elicit significant changes in Th2 cell or Treg cell responses to peanut, although there were trends for elevation in both.

Taken together these results indicate that IgE antibodies promote immune sensitization to ingested allergens. In the food allergy susceptible mice harboring a dysregulated form of IL-4rα, the presence of IgE during recurrent enteral peanut exposure promoted the induction of peanut-specific Th2 cell, IgE and IgG1 responses while suppressing the expansion of anti-allergic Treg cells. Moreover, IgE blockade in animals with established allergy served to reverse sensitivity with a reduction in Th2 cell responses and expansion of Treg cells.

Mast cells amplify allergic responses to ingested peanut and suppress peanut-specific Treg cell induction

We hypothesized that mast cells were the likely effectors of IgE-mediated Th2 cell-induction and Treg cell-suppression observed in the peanut allergy model. In order to test this, we bred the F709 IL-4rα mutation onto the C57BL/6 genetic background and then intercrossed these mice with KitW-sh mice. These mice mast cell-deficient exhibited a dramatic (3-log) reduction in peanut-specific IgE along with significantly impaired Th2 cell responses and markedly enhanced Foxp3+ Treg cell induction (Figure 3A, B, C, Figure S2F). Reconstitution of the mast cell-deficient mice with WT bone marrow-derived mast cells (BMMC) restored Th2 cell responses while partially but significantly restoring IgE production (Figure 3A, B). Mast cell-reconstituted mice exhibited impaired Treg cell generation (Figure 3C). Unlike their mast cell-sufficient counterparts, mast cell-deficient mice subjected to peanut sensitization were protected from anaphylaxis, but anaphylaxis was restored by mast cell reconstitution (Figure 3D). In contrast to WT BMMC-reconstituted mice, mice reconstituted with IL-4-deficient mast cells failed to develop peanut-specific IgE, Th2 or anaphylactic responses, instead showing enhanced Treg cell induction (Figure 3). Mast cell reconstitution of the small intestine of mast cell-deficient mice was confirmed by histologic examination (Figure S3).

Figure 3. Mast cells are required for sensitization to food allergens.

Figure 3

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A) PN-specific IgE levels in sera as determined by ELISA. IL-4rα F709 mutant (Il4ratm3.1Tch) and mast cell-deficient Il4ratm3.1Tch KitW-sh mice (n=6–8) were sensitized by enteral gavage with 23mg PN once a week for eight weeks. Mast cells were reconstituted in additional Il4ratm3.1Tch KitW-sh mice by i.p. injection of WT or IL-4-deficient (Il4tm1Cgn) BMMC (5×106) at four and eight weeks prior to sensitization. Kit variants are abbreviated as “+” for WT and “−” for KitW-sh. B) IL-4+ CD4 T cells in the MLN. C) Foxp3+ Treg cell frequencies in CD3ε+CD4+ MLN cells exhibiting proliferative responses to PN. D) Temperature curves following acute enteral challenge with PN (450mg) after sensitization of IL-4rα F709 mutant (Il4ratm3.1Tch). P<0.001 by RM two-way ANOVA Kit+/+ vs. KitW-sh, KitW-sh vs. KitW-sh +BMMC and KitW-sh +BMMC vs. KitW-sh +Il4tm1Cgn BMMC. Data shown are from one of two experiments, and are depicted as mean ±SEM.

Although KitW-sh mice exhibit complete mast cell deficiency and are amenable to reconstitution using BMMC, making them an attractive model system in which to study mast cell biology, they have an array of immunological defects including dysregulation of granulocytes introducing potential complications to the interpretation of our results. In order to further evaluate mast cell functions in the peanut allergy model we took advantage of the Kit-independent Mcpt5cre transgene system to genetically target mast cells. A two-pronged approach was applied that used Mcpt5 promoter-driven Cre expression either to mediate mast cell deletion via induction of iDTR followed by treatment with diphtheria toxin (Il4ratm3.1Tch Mcpt5cre iDTR) or to drive mast cell-specific excision of the Sykb gene (Il4ratm3.1Tch Mcpt5cre Sykfl/fl). We reasoned that removing Syk, the proximal kinase in FcεRI signaling, would paralyze IgE-mediated mast cell activation allowing us to isolate the role of IgE-mediated mast cell activation within this model (Figures S4, S5).

Mice in which these genetic approaches were used to achieve mast cell ablation or mast cell paralysis behaved similarly, exhibiting both a restoration of oral tolerance and reduction in the peanut allergic phenotype. Specific IgE and Th2 responses were reduced in these mice relative to controls (Figure 4A and B). As we had observed using the KitW-sh model, removal of mast cells was sufficient to restore Treg cell generation to near wild-type levels even in the face of the pro-allergic IL-4rα F709 mutation (Figure 4C). Anaphylaxis was undetectable in peanut-sensitized Il4ratm3.1Tch Mcpt5cre iDTR mice that receive DT treatment prior to sensitization, and similarly absent in peanut-sensitized Il4ratm3.1Tch Mcpt5cre Sykfl/fl mice (Figure 4D). Strikingly, deletion of Syk solely from mast cells was sufficient to restore Treg cell induction, identifying IgE crosslinking of FcεRI on mast cells as the key signal tipping the balance between allergy and tolerance following food allergen exposure in an environment genetically permissive for the induction of food allergy.

Figure 4. Deletion of Syk in mast cells prevents peanut sensitization.

Figure 4

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A) PN-specific IgE levels in sera. Mast cell-directed induction of the diphtheria toxin receptor (DTR) or inactivation of Syk tyrosine kinase on the IL-4rα F709 (Il4ratm3.1Tch) background were achieved using Mctp5cre-driven gene expression. Mice expressing iDTR on mast cells (Il4ratm3.1Tch Mcpt5cre iDTR) or with mast cell-targeted Syk deletion (Il4ratm3.1Tch Mcpt5cre Sykfl/fl) (n=6–11) were sensitized once a week for four weeks with 23mg PN i.g. Mast cells were depleted from Mcpt5cre iDTR mice by i.p. injection of diphtheria toxin over three days (100ng, 500ng, 500ng) one week prior to initiating PN sensitization (indicated as iDTR DT “+”). B) ELISA analysis of PN-specific IL-4 secretion in splenocyte cultures. C) Foxp3+ Treg cell frequencies among PN-responding CD3ε+CD4+ T cells from the MLN. D) Temperature curves from PN-treated Il4ratm3.1Tch mice following enteral challenge with high-dose PN (450mg). P<0.001 by RM two-way ANOVA Mcpt5cre vs. Mcpt5cre iDTR and Mcpt5cre vs. Mcpt5cre Sykfl/fl). Data shown are a pool of three independent experiments, represented as mean ±SEM.

In summary, these observations demonstrate that allergic sensitization (Th2 cell responses and IgE production) are reduced and Treg cell responses enhanced in two independent strains of mice harboring the activating IL-4rα mutation but lacking mast cells and in mice in which FcεRI signaling is impaired specifically in mast cells. Repletion of the mast cell compartment by wild-type but not IL-4-deficient BMMC transfer restores the food allergy-prone phenotype of the IL-4rα mutant mice, confirming a direct role for mast cells and IL-4 produced by them in driving allergic sensitization and suppressing tolerance.

Syk-blockade prevents allergic sensitization in naïve mice and facilitates induction of tolerance in food-allergic animals

As the key kinase in FcεRI receptor signaling, Syk represents an attractive target for pharmacologic inhibition in the treatment of allergic disease. We have recently characterized a new highly specific, potent, orally bioavailable Syk inhibitor, SYKi and applied this to probe the consequences of FcεRI blockade in the peanut model (Moy et al., 2013). SYKi displayed a dose-dependent inhibition of passive IgE-mediated anaphylaxis and completely abrogated the lethal anaphylactic reaction to acute food allergen challenge in sensitized IL-4rα mutant mice (Figure S6). We anticipated that inhibition of Syk would block sensitization to food allergens, much the way that sensitization was blocked in mice with targeted genetic deletion of Syk in mast cells. This proved to be the case. Peanut-specific IgE responses were very low or undetectable in mice treated with SYKi during sensitization, and T cell responses correspondingly shifted from Th2 cell-biased to Treg cell-dominated (Figure 5A–C). Peanut-specific IgG1 responses remained largely intact in SYKi-treated mice, indicating that the absence of IgE did not result from a global suppression of B cell function. However we cannot rule out effects on Syk-mediated B cell receptor signaling using this compound (Figure 5D). Treatment with SYKi during sensitization was sufficient to prevent the development of anaphylactic food allergy to peanut, limiting temperature loss and preventing MMCP-1 release (Figure 5E, F). To ensure complete clearance of SYKi, peanut challenge was performed after a ten-day “wash-out” period.

Figure 5. Targeted pharmacologic inhibition of Syk blocks food allergen sensitization.

Figure 5

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A) PN-specific IgE levels in sera as measured by ELISA. IL-4rα F709 (Il4ratm3.1Tch) mutant mice (n=12–15) were sensitized once a week by enteral gavage with PN (23mg) for four weeks. Mice were also gavaged with the Syk inhibitor SYKi (30mg/kg) or vehicle (10% Tween-80, pH 8.0, 5ml/kg) 15min prior to PN, 10hr post-PN and 24hr post-PN. PN: PN plus vehicle during sensitization; PN+SYKi: PN plus SYKi during sensitization. B) Flow cytometric analysis of IL-4 production by expanded CD4+ MLN cells exhibiting proliferative responses to PN in vitro (n=5). C) Frequency of Foxp3+ cells among CD3ε+CD4+ T cells proliferating to PN in vitro (n=12–15). D) Serum levels of PN-specific IgG1 after sensitization, as determined by ELISA. E) Degranulatory release of MMCP-1 into serum following sensitization and challenge with PN (n=10–11). There was a ten-day interval between the final SYKi treatment and the PN challenge. F) Temperature curves following acute challenge with PN (450mg) in PN+vehicle-, PN+SYKi- or saline-treated mice (n=5). P<0.001 by RM two-way ANOVA PN vs. PN+SYKi. Data in B and F are representative of two experiments each, while the data in panels A, C, D and E have been pooled from three independent experiments. Data are shown as points for individual mice with mean ±SEM.

In order to test whether Syk-inhibition, like anti-IgE treatment, might facilitate tolerance induction in peanut-allergic animals, mice were sensitized to peanut and then separated into groups that received 1) no further treatment, 2) high-dose peanut desensitization or 3) high-dose daily peanut under cover of SYKi. Mice were evaluated ten days after the discontinuation of therapy. Mice subjected to peanut sensitization and those with peanut-only desensitization therapy developed normal anaphylactic reactions, the SYKi plus peanut group tolerated the challenge and displayed no symptoms of anaphylaxis (Figure 6A and Figure S7). MMCP-1 release was reduced by approximately 2 logs (Figure 6B). Peanut-specific Th2 cell responses were nearly absent in the tolerized mice, and specific Treg cell frequencies increased (Figure 6C, D). Peanut-specific IgE was reduced in mice receiving SYKi plus peanut compared with mice receiving peanut-only desensitization (Figure 6E). Our data demonstrate that the addition of SYKi to peanut desensitization markedly altered several critical aspects of the food allergen immune response in a manner consistent with tolerance induction (Figure S7).

Figure 6. Pharmacological inhibition of Syk activity enhances food allergen desensitization.

Figure 6

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A) Temperature curves following acute challenge with PN in PN-sensitized (PN), PN+SYKi-desensitized (PN, PN+SYKi) and mock-desensitized (PN, PN) mice (n=5). PN-sensitized IL-4rα F709 (Il4ratm3.1Tch) mutant mice were subjected to three weeks of daily oral desensitization therapy with PN (225mg) (PN, PN), or PN paired with SYKi (30mg/kg, p.o., b.i.d.) (PN, PN+SYKi). The final challenge was performed in the absence of SYKi, ten days after the final SYKi dose. PN-sensitized mice that received no therapy after sensitization are shown for comparison (PN). P=0.0016 by RM two-way ANOVA for PN vs. PN, PN+SYKi or PN, PN vs PN, PN+SYKi. B) MMCP-1 levels in serum post-challenge (n=4–5). C) IL-4 secretion by splenocyte cultures in response to PN-stimulation (n=9–10). D) PN-specific Treg cell frequencies in dividing MLN CD3ε+CD4+ cells (n=5–6). E) Serum levels of peanut-specific IgE (n=9–10). Data shown are representative of two independent experiments (A, B and D) or have been pooled from two experiments (C and E), and are represented as mean ±SEM.

Tolerance to foods is maintained in part by Treg cells and Treg cell deficiency is associated with severe food allergy (Bennett et al., 2001b; Chatila et al., 2000b; Wildin et al., 2001b). Our analysis of Foxp3+ T cell frequencies in both the peanut and OVA food allergy models suggested that the mechanism underlying peanut desensitization in the setting of IgE:FcεRI signaling blockade was the induction of functional Treg cell, capable of suppressing effector T cell responses. This potential mechanism was directly evaluated by preparing Foxp3-eGFP+ Treg cells from mice that had been sensitized to peanut in the presence or absence of SYKi, and transferring these cells into naïve Il4ratm3.1Tch recipients, which were in turn sensitized to peanut (Figure 7A). Treg cells from peanut+SYKi-treated donors almost completely ablated specific IgE responses in peanut-fed recipients (Figure 7B). T cell responses in the same mice exhibited significant shifts from Th2 cell- to Treg cell-biased (Figure 7C, D and Figure S2G, H). Only mice given Treg cells from peanut+SYKi-treated mice exhibited reductions in anaphylaxis and MMCP-1 release upon peanut challenge (Figure 7E, F). Recipients of Treg cells from mice exposed to peanut in the absence of SYKi had much more modest (and only significant in the case of IgE) trends relative to mice that did not receive Treg cells.

Figure 7. Treg cells induced while mast cells are paralyzed effectively control food allergen sensitization.

Figure 7

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A) Experimental schematic. IL-4rα F709 mutant Thy1.1-congenic Foxp3-eGFP (Il4ratm3.1Tch Foxp3tm2Tch Thy1a) donor mice (n=5) were sensitized to peanut with or without SYKi for four weeks, at which point their CD4+Foxp3-eGFP+ Treg cells (TR) were purified and transferred (4×105/mouse) into naïve IL-4rα F709 (Il4ratm3.1Tch) recipients (for PN and PN+SYKi TR groups). Mice that did not receive Treg cells are included as positive controls for peanut sensitization. All recipient mice were sensitized with 23mg PN once a week for four weeks and challenged with 450mg PN by gavage (n=5). B) PN-specific IgE production in recipient mice. C) Flow cytometric analysis of IL-4 in CD4+ T cells from the MLN. D) Foxp3+ Treg cell frequencies in the MLN of recipient mice as assessed by flow cytometric analysis of CD4+CD3ε+ cells taken directly ex vivo. Data in C and D exclude Thy1.1+ donor cells from the analyses. E) Serum MMCP-1 release following acute enteral challenge of recipient mice with PN (450mg) F) Core temperature loss following PN challenge in recipient mice (P<0.001 by RM two-way ANOVA No TR vs. PN+SYKi TR and PN+SYKi TR vs. PN TR). The skull-and-crossbones symbol indicates a death from anaphylaxis in the PN TR group. Data are from a single experiment, and are shown as mean ±SEM.

Taken together, these results show that pharmacologic blockade of FcεRI signaling using a Syk inhibitor prevents food allergen sensitization and promotes tolerance induction in food allergy-prone IL-4rα mutant mice. The Treg cell skewing effect of Syk inhibition can be exploited to restore tolerance in mice with established allergy. Our observations of food allergy suppression in recipients of Treg cells confirm the role of functional peanut-specific Foxp3+ Treg cells in oral tolerance. SYKi has no activity in T cells and in the context of our corroborating data using mice with lineage specific Syk deletion in mast cells, mast cell deficiency models and anti-IgE treatment we attribute the block in functional Treg cell induction to FcεRI signaling blockade rather than any T cell intrinsic effect.

DISCUSSION

Mast cells reside at the interfaces between the body and the environment, stationing them as sentinels for the immune system. IgE antibodies arm these innate immune cells for specific antigen recognition. In the setting of food allergy, our findings suggest a triple function for these first-line defenders: 1) as effectors of anaphylactic reactions in allergen exposed subjects, 2) as amplifiers of nascent Th2 cell and IgE responses during recurring sensitizing ingestions of food allergen and 3) as suppressors of functional Foxp3+ Treg cell induction. Our report shows that mast cells and IgE:FcεRI signaling are required for the induction of adaptive Th2 cell immunity to ingested allergens and that they counter Treg cell expansion; in the absence of mast cells or IgE signals, peanut fed allergy-prone mice exhibited minimal Th2 cell and IgE responses but had robust Treg cell induction. This work was carried out using an innovative array of research tools including the novel IL-4rα mutant model of peanut allergy, in which enteral ingestion alone leads to anaphylactic food sensitivity, and a potent selective Syk inhibitor to modulate IgE:FcεRI signaling. The observation that inhibition of IgE signaling with anti-IgE antibody or Syk inhibition in the setting of pre-existing allergy leads to reversal of IgE and Th2 cell responses along with Treg cell expansion is completely new and quite striking. This result reveals significant plasticity in food allergy responses and points to Syk as an attractive therapeutic target in food allergy.

Food allergy has been notoriously difficult to model in mice. Even with the use of adjuvants and parenteral priming, which bypass normal immune sensitization mechanisms, responses to food challenge are weak. Challenged animals do not exhibit anaphylaxis, a cardinal symptom of food allergy in patients. The IL-4rα mutant mice have provided a powerful genetic tool to address the pathogenesis of food allergy in a more physiologic setting. We believe the inherent predisposition to allergy, based on enhanced IL-4rα responses, in these mice reflects a cytokine-signaling imbalance that might be at work in atopic humans. We have previously reported that an IL-4rα polymorphism affecting signaling strength is linked to asthma (Hershey et al., 1997). In studies designed to identify loci linked with IgE production, the chromosome 5q31 cytokine cluster, which contains the gene encoding IL-4, is consistently identified (Marsh et al., 1994; Meyers et al., 1994; Xu et al., 1995) and one preliminary study reported a significant association between an IL4Ra polymorphism and food allergen-specific IgE levels (Brown et al., 2012). We believe that deletion of the ITIM in the IL-4Rα of the mice we have studied and the resultant disinhibition of the receptor are paradigmatic of the situation in atopic humans in whom nascent allergic sensitization also appears to be affected by an altered IL-4 signaling axis.

We and others have previously demonstrated that IgE antibodies exert important effects on mast cells above and beyond the induction of degranulation and immediate hypersensitivity. Even in the absence of antigen, IgE regulates mast cell homeostasis in culture and in vivo and modulates mast cell cytokine production (Asai et al., 2001; Bryce et al., 2004; Kalesnikoff et al., 2001; Kawakami and Kitaura, 2005; Mathias et al., 2009). Similar effects on mast cell cytokine production are observed in systems involving antigen-driven IgE signaling as well (Galli et al., 2005; Nakae et al., 2007a). The effects of IgE-mediated mast cell signaling in modulating Th2 cell and Treg cell responses in the peanut allergy system in this report are corroborated by four independent lines of investigation: the study of IgE-deficient mice, anti-IgE treatment, Mcpt5cre Sykfl/fl mice and SYKi-treatment. Each of these experimental models demonstrated impairment of Th2 cell and IgE responses along with enhanced Treg cell induction, providing a strong aggregate case for a Th2 cell adjuvant effect of IgE signaling in emerging food allergy responses. These results have potential implications for the design of strategies to achieve safe and effective oral desensitization in patients with peanut allergy. Although anti-IgE treatment during food desensitization has recently been applied in clinical trials the immunomodulatory effects of such IgE-blockade are not yet known.

The role of mast cells in murine disease models is challenging to assess given the limitations of each of the various mast cell deficiency models (Rodewald and Feyerabend, 2012). The fully consistent results we independently obtained using both Kit-dependent and -independent models of deficiency (KitW-sh and Mcpt5cre iDTR respectively) provide strong converging support for a critical mast cell contribution to Th2 cell induction and Treg cell suppression. Whereas Mcpt5cre iDTR mice have previously been reported to have an intact mucosal mast cell compartment (based on baseline histologic analyses of the stomach), we have found that IL-4-driven and allergen-induced mucosal mast cell expansion in the jejunum is in fact suppressed in these animals, and it has been reported that MMCP-5 expression is not restricted to submucosal mast cells under Th2 conditions (Friend et al., 1996; Xing et al., 2011). DT-treated animals exhibited both impaired mast cell expansion and the same block in Th2 cell and IgE responses observed in IgE-deficient and KitW-sh mutants.

Although mast cells are not MHC II+ and may not function as antigen presenting cells in this food allergy model system, we observed that they accumulate in the draining lymph nodes of sensitized animals, placing them proximal to T cell priming events. By immunofluorescent microscopy, both Treg cells and mast cells are present in the jejunal lamina propria and Peyer’s patches (data not shown). Mast cells are known to produce significant amounts of the critical Th2 cell-inducing cytokine, IL-4, which has the capacity both to subvert Treg cell generation, by destabilizing Foxp3 expression, while simultaneously activating the Th2 and Th9 pathways via STAT6 signals (Dardalhon et al., 2008; Wang et al., 2010). We observe that when mast cells are cultured with naïve T cells under Treg cell-inducing conditions, FcεRI crosslinking inhibits TGFβ-driven expansion of Foxp3+ T cells. Furthermore, we find that Il4 and Il13 transcripts are very rapidly induced (within 1hr) in the small intestine following allergen exposure and that these transcripts are Syk- and IgE-dependent, strongly implicating mast cells as cellular sources of IL-4 and IL-13 early after food ingestion. Our results from WT vs. IL-4-deficient mast cell reconstitution of mice lacking mast cells demonstrate an essential role for mast cell-derived IL-4 in promoting sensitization to ingested allergen.

Our findings that 1) peanut-specific Treg cell frequencies are higher in IgE-deficient, anti-IgE-treated, mast cell-deficient, mast cell Syk-deficient and SYKi-treated mice and that 2) Treg cells from peanut-fed SYKi-treated mice functionally suppress the induction of peanut allergy in naïve recipients identify a central mechanism whereby IgE antibodies and mast cells enhance emerging allergic sensitization: they suppress effective Treg cell responses. We and others have previously demonstrated that inherited Foxp3 deficiency leads to severe intractable food allergy and that patients with Foxp3 mutations lack functional Treg cells (Bennett et al., 2001a; Chatila et al., 2000a; Wildin et al., 2001a).

Syk inhibition offers an attractive pharmacologic approach for blocking IgE-induced FcεRI signaling. Previously available small molecule Syk inhibitors have had significant off-target effects, however, especially on the closely related kinase, ZAP70, in T cells. SYKi, used in this study has >25-fold enhanced specificity for fully phosphorylated Syk vs. ZAP70 (Moy et al., 2013). Although Syk is also the proximal signaling kinase for the B-cell receptor (BCR), we believe that its major effects on allergic responses to peanut in our study resulted from its FcεRI inhibition. In support of this conclusion is our finding that the phenotype of peanut-fed mice in which Syk is specifically inactivated in mast cells but not B cells, exactly mirrors that of the SYKi-treated animals. BCR activation is normal in the mice with mast cell-targeted Syk gene deletion yet they exhibited the same impairment in Th2 cell and IgE induction and enhanced Treg cell responses to SYKi treated mice consistent with an FcεRI-driven effect.

In summary, our study provides clear evidence that IgE-mediated mast cell activation serves not only to elicit immediate hypersensitivity reactions but also to amplify evolving allergic sensitization to ingested proteins. We speculate that manipulations of the IgE axis may some day provide clinical benefit.

EXPERIMENTAL PROCEDURES

Mice

Detailed mouse information can be found in the Supplemental Material. All mice were bred and maintained under specific pathogen-free conditions at Boston Children’s Hospital. All animal experiments were conducted under procedures approved by the Boston Children’s Hospital Institutional Animal Care and Use Committee.

Allergic sensitization and anaphylaxis

Mice were sensitized by enteral gavage with ball-tipped 18g feeding needles. Peanut butter (Skippy, Hormel Foods) was gavaged in 250μl 0.1M sodium bicarbonate pH 8.0. Mast cells were depleted by i.p. injection of diphtheria toxin (List Biological Laboratories) in saline over three days: 100ng day 1, 500ng day 2 and 500ng day 3. SYKi was prepared as previously described (Moy et al., 2013) and solubilized in pH 8.0 aqueous isosmotic Tween-80 (10% w/v, Sigma). SYKi (30mg/kg) and vehicle control were delivered by gavage in 5ml/kg. Anti-IgE (clone R35-92, BD) was injected i.p. in tripling doses every hour starting at 50ng, integrating to a total of 25μg the first day. Over the next three days, 25μg anti-IgE was injected once a day i.p., giving a total of 100μg anti-IgE per mouse. Core body temperature measurements taken to monitor anaphylaxis were recorded using sub-dermally implanted transponders (IPTT-300) and a DAS-6001 console (Bio Medic Data Systems) linked to a netbook. For sensitization protocols and further information, see Supplemental Material (Figure S1).

Cell culture

Bone marrow-derived mast cells (BMMC) were cultured as previously described (Burton et al., 2013). BMMC were used for reconstitution experiments after 3–6 weeks of culture. Splenocytes were cultured in RPMI-1640 supplemented as previously described (Burton et al., 2013), and stimulated for antigen-specific recall with 100μg/ml peanut extract. Endogenous allergen-specific T cells were identified by allergen-induced ex vivo proliferation, along the lines of assays developed for assessing human tetanus toxoid recall (Narendran et al., 2002). MLN cells (5×106) were labeled with Violet CellTrace (Life Technologies) and cultured with or without allergen (100μg/ml peanut extract) for five days. Cells undergoing proliferation (dye dilution) were considered allergen-specific, a conclusion supported by a lack of proliferation in the absence of allergen or in allergen-stimulated cells from unsensitized mice. We note that this approach does not provide the absolute number of Treg cells residing in the MLN but nevertheless gives a robust readout of the relative efficiency with which Treg cells are induced among experimental groups.

Mast cell reconstitution

Weanling Il4ratm3.1Tch KitW-sh mice were injected i.p. with 5×106 BMMC and a second injection of BMMC was performed four weeks later. Allergic sensitization was started eight weeks after the initial transfer, at which point mast cell reconstitution of intestinal compartments has been shown to be evident (Grimbaldeston et al., 2005). Reconstitution was performed using matched cultures of WT C57BL/6J and IL-4-deficient B6.129P2-Il4tm1Cgn/J (stock number 002253) mast cells prepared from age-matched female mice purchased from Jackson Laboratories (Bar Harbor, ME).

Flow cytometry, cell sorting and ELISA

See Supplemental Material.

Histology

Sections of the jejunum were taken approximately 10cm from the pyloric sphincter and fixed overnight in 10% formalin (Sigma). Samples were stored in 70% ethanol prior to processing and toluidine blue staining by the Beth Israel Deaconess Medical Center Histology Core. Chloroacetate esterase staining, which stains mast cells with a characteristic red color, was performed as previously described (Burton et al., 2013; Friend et al., 1998). Samples were randomized and coded prior to histological processing, with scoring and staining being performed by an investigator unaware of the sample identities.

Statistics and data analysis

Data were graphed and analyzed using GraphPad Prism 5.0. Anaphylaxis temperature curves were analyzed using repeated measures two-way ANOVA for overall distinction between treatment groups. Unpaired t tests were used to compute two-tailed P values for comparisons between two groups. For three or more groups, ANOVA was used, with individual P values coming from Bonferroni post-tests. P values are abbreviated as *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Since peanut-specific IgE and MMCP-1 levels varied by a few orders of magnitude, these data are presented on graphs with logarithmic Y-axes, and were log-transformed prior to statistical analysis. Samples in which specific immunoglobulins or MMCP-1 were undetectable were assigned a nominal value corresponding to the limit of detection in the assay (0.3125 units/ml for peanut-specific IgE). Data are presented as mean ±SEM, with points representing individual mice where presented.

Supplementary Material

01

HIGHLIGHTS.

  • Mice with a disinhibiting mutation of the IL-4 receptor can develop peanut allergy.

  • Antibody and cellular responses to peanut are enhanced by mast cells and IgE.

  • IgE-inhibition blocks induction of peanut allergy and promotes Treg cell induction.

  • Syk blockade both prevents allergic sensitization and reverses established allergy.

Acknowledgments

This work was supported by NIH NIAID grants: R01 AI085090 (TAC), R56 AI100889 (HCO), T32 AI007512 (OTB, SLL), P30 DK034854, the Boston Children’s Hospital Translational Research Program (HCO), and by the Bunning Foundation (HCO, TAC). Dr. Houshyar is an employee of Merck and Co. as was Dr. Crackower at the time the study was performed.

Footnotes

AUTHOR CONTRIBUTIONS

OTB, HCO, MNR and TAC designed experiments. OTB and MNR performed experiments and developed experimental models. SLL and JSZ helped carry out experiments. KJK and ARD provided technical assistance and developed assays. HH and MAC participated in the experimental design and data analysis for the SYKi experiments. AR provided Mcpt5cre mice and participated in the analysis of the Mcpt5cre data. OTB and HCO wrote the manuscript. All authors participated in discussions of experimental results and edited the manuscript.

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Supplementary Materials

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NIHMS608517-supplement-01.pdf (5MB, pdf)

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