Mast Cell Responses in a Mouse Model of Food Allergy are Regulated via a ST2/IL-4 axis

To the Editor,

Allergen-induced mast cell (MC) activation is critical for the development of food allergy. Recent evidence implicates the alarmin cytokine, IL-33, as a key player in regulating MC responses during allergic inflammation. The IL-33 receptor, ST2, is constitutively expressed on MCs, and genetic polymorphisms within the IL-33/ST2 axis are strongly linked to disease susceptibility¹. Several recent studies have demonstrated a critical role for IL-33 in inducing MC responses to food antigens, including decreased MC activation and food allergy development in both epicutaneously-sensitized ST2^−/− mice^2,3 and mice treated with anti-IL-33 antibodies^2,4. Similarly, IL-33-stimulated group 2 innate lymphoid cells can also activate MCs in enterally-challenged mice⁵. However, depending on the model of allergic sensitization (epicutaneous – with or without tape-stripping), divergent effects on MC activation, intestinal MC numbers, and anaphylaxis have been observed^2,3. While epicutaneously-sensitized, tape-stripped ST2^−/− mice were protected from anaphylaxis due to decreased MC activation, no changes in intestinal MC expansion were observed². In contrast, in non-tape-stripped ST2^−/− mice, both MC activation and intestinal MC numbers were decreased³.

We sought to investigate the role of IL-33 using a well-established model of systemic sensitization to food antigens. Briefly, wild-type Balb/c (WT) and ST2^−/− mice were immunized i.p. with chicken egg ovalbumin (OVA), followed by intragastric OVA challenges. Oral allergen exposure resulted in profuse allergic diarrhea in sensitized WT mice (Fig. 1A), accompanied by a robust antigen-mediated IgE response (Fig. 1B), significant MC accumulation in the jejunum (Fig. 1C), and increased MC activation (Fig. 1D), as indicated by elevated serum MC protease-1 (MCPT-1). In contrast, ST2^−/− mice exhibited decreased diarrhea and MC responses (Figs. 1A–D). Similarly, the expression of the type 2 cytokine genes, IL-4 and IL-13, was also reduced in the intestines of ST2^−/− mice (Figs. 1E–F). In contrast, enhanced expression of IL-33 was observed in OVA-sensitized ST2^−/− mice (Fig. S1 A). Similarly, a trend towards increased expression of IFN-γ was also observed (Fig. S1 B), suggesting that a decrease in type 2 cytokines may be accompanied by a parallel increase in type 1 cytokines. IL-33 is critical for the induction of allergen-specific Th2 cells^6,7. We therefore wondered whether systemic T cell responses may also be impaired in the absence of ST2. As observed in Figs. 1G–J, while splenocytes from OVA-challenged WT mice secreted elevated levels of IL-4, IL-5 and IL-13 in response to antigen restimulation, no such enhancement was observed in the ST2^−/− group. Similarly, T cell receptor-mediated polyclonally-induced type 2 cytokines were also decreased in OVA-challenged ST2^−/− mice (Fig. S1 C–F).

Figure 1. Mast cell responses and food allergy are attenuated in OVA-challenged ST2^−/− mice.

WT and ST2^−/− mice were sensitized and challenged with OVA as described in Supplemental Methods. One hour after the final challenge, mice were sacrificed, and the development of food allergy was assessed. (A) Percent of animals with diarrhea (B) Serum OVA-IgE (C) Jejunal mast cells (D) Serum MCPT-1 (E-F) Jejunal expression of IL-4 and IL-13 and (G-J) Expression of IL-4, IL5, IL-13 and IFN-γ following ex vivo stimulation of splenocytes by OVA for 72 hours. n=4–8 mice per group. Data are representative of 5 independent experiments. ND = not detected. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (Student’s t-test).

Considering the surprisingly low levels of T cell-derived IL-4 in the absence of IL-33 signaling and given that IL-4 has been shown to induce MC activation⁸, we wondered whether the attenuated MC responses in OVA-sensitized ST2^−/− mice may be a consequence of decreased IL-4 production. This is consistent with previous studies demonstrating decreased IL-4 production in antigen-specific T cells from ST2^−/− mice⁹. Furthermore, while we did not observe any differences in baseline IL-4 production from polyclonally stimulated naïve WT or ST2^−/− splenic T cells (Fig. S2 A and as has also been observed previously⁹), IL-4Rα expression was decreased in both bone marrow-derived MCs (Fig. S2 B) and OVA-sensitized CD4 T cells (Fig. S2 C) from ST2^−/− mice. Lastly, the induction of food allergy in this model has also been shown to be dependent on IL-4 with both antibody-mediated depletion and genetic ablation resulting in the suppression of MC responses¹⁰. To therefore further assess the role of IL-4, we treated mice with IL-4C to boost IL-4 levels in ST2^−/− mice. To our surprise, IL-4 treatment not only amplified the effects of allergen challenge in WT animals, but completely reversed it in ST2^−/− mice, resulting in a significant increase in OVA-IgE and serum MCPT-1 (Figs. 2A&B). Furthermore, while all of the allergen-challenged ST2^−/−+IL-4-treated mice developed diarrhea, several animals also died from anaphylactic shock (Fig. 2C and data not shown). Finally, IL-4 treatment also resulted in enhanced intestinal MC accumulation in allergen-challenged ST2^−/− mice (Figs. 2D&E).

Figure 2. IL-4 treatment reverses mast cell activation and food allergy development in ST2^−/− mice.

WT and ST2^−/− mice were sensitized and challenged with OVA as described in Supplemental Methods. Some groups of mice received IL-4C prior to both OVA sensitization and each OVA challenge. One hour after the final challenge, mice were sacrificed, and the development of food allergy was assessed. (A) Serum OVA-IgE (B) Serum MCPT-1 (C) Percent of animals with diarrhea and (D-E) Jejunal mast cells are shown. n=6–7 mice per group. Data are representative of 2 independent experiments. ND = not detected. *p<0.05; **p<0.01; ***p<0.001 (Student’s t-test).

Collectively, our data demonstrate that IL-33 plays a critical role in mediating MC responses to food allergic sensitization, with effects on both MC expansion and activation. Furthermore, these effects are mediated via induction of IL-4, resulting in restoration of MC responses in ST2^−/− mice. Treatment with dupilumab has become a staple of immunotherapy for several allergic diseases. Considering that IL-33 neutralization is also a current target of investigation in clinical trials¹¹, our data shed further light on the potential interplay between these cytokines during treatment with these regimens.