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. 2016 Sep;138(3):801-811.e9.
doi: 10.1016/j.jaci.2016.02.030. Epub 2016 Apr 18.

IL-4 production by group 2 innate lymphoid cells promotes food allergy by blocking regulatory T-cell function

Magali Noval Rivas  1 , Oliver T Burton  1 , Hans C Oettgen  1 , Talal Chatila  2
Affiliations

IL-4 production by group 2 innate lymphoid cells promotes food allergy by blocking regulatory T-cell function

Magali Noval Rivas et al. J Allergy Clin Immunol. 2016 Sep.

Abstract

Background: Food allergy is a major health issue, but its pathogenesis remains obscure. Group 2 innate lymphoid cells (ILC2s) promote allergic inflammation. However their role in food allergy is largely unknown.

Objective: We sought to investigate the role of ILC2s in food allergy.

Methods: Food allergy-prone mice with a gain-of-function mutation in the IL-4 receptor α chain (Il4raF709) were orally sensitized with food allergens, and the ILC2 compartment was analyzed. The requirement for ILC2s in food allergy was investigated by using Il4raF709, IL-33 receptor-deficient (Il1rl1(-/-)), IL-13-deficient (Il13(-/-)), and IL-4-deficient (Il4(-/-)) mice and by adoptive transfer of in vitro-expanded ILC2s. Direct effects of ILC2s on regulatory T (Treg) cells and mast cells were analyzed in coculture experiments. Treg cell control of ILC2s was assessed in vitro and in vivo.

Results: Il4raF709 mice with food allergy exhibit increased numbers of ILC2s. IL-4 secretion by ILC2s contributes to the allergic response by reducing allergen-specific Treg cell and activating mast cell counts. IL-33 receptor deficiency in Il4raF709 Il1rl1(-/-) mice protects against allergen sensitization and anaphylaxis while reducing ILC2 induction. Adoptive transfer of wild-type and Il13(-/-) but not Il4(-/-) ILC2s restored sensitization in Il4raF709 Il1rl1(-/-) mice. Treg cells suppress ILC2s in vitro and in vivo.

Conclusion: IL-4 production by IL-33-stimulated ILC2s blocks the generation of allergen-specific Treg cells and favors food allergy. Strategies to block ILC2 activation or the IL-33/IL-33 receptor pathway can lead to innovative therapies in the treatment of food allergy.

Keywords: Anaphylaxis; IL-13; IL-33; IL-4; food allergy; group 2 innate lymphoid cells; innate lymphoid cells; mast cells; nuocytes; oral tolerance; regulatory T cells.

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Figures

Figure 1
Figure 1. ILC2 are enriched in peanut-sensitized Il4raF709 mice
(A) Core body temperature changes in PBS or peanut-sensitized WT and Il4raF709 mice after oral peanut challenge. (B) Serum concentrations of peanut-specific IgE and MMCP-1 after anaphylaxis. (C) Il33 and Il25 mRNA levels in the SI of PBS and Peanut-sensitized WT and Il4raF709 mice. (D – E) Flow cytometric analysis (D), frequencies and absolute numbers (E) of ILC2 (Lin− CD25+ CD127+ Sca-1high c-Kitlow) and ILC3 (Lin− CD25+ CD127+ Sca-1− c-Kit+) in the MLN. (F and G), Frequencies and absolute numbers of IL-13+ ILC2 and IL-17+ ILC3. Results represent data on 3 to 15 mice per group derived from two independent experiments. *P < .05, **P < 0.01 and ***P < 0.001 by two-way ANOVA and one-way ANOVA with post-test analysis.
Figure 2
Figure 2. Il1rl1 signaling drives ILC2 expansion and peanut allergy
(A) Core body temperature change in peanut-sensitized WT, Il1rl1−/−, Il4raF709 and Il4raF709 Il1rl1−/− mice after oral challenge. (B) Serum concentrations of peanut-specific IgE and MMCP-1 after anaphylaxis. (C –D) Frequencies and absolute numbers of ILC2 and ILC3 in the MLN (C) and SI (D) from the mouse groups in (A). Results represent 3 to 8 mice per group derived from two independent experiments; *P < .05, **P < .01 and ***P < .001 by two-way ANOVA and one-way ANOVA with post-test analysis.
Figure 3
Figure 3. Th2 cytokine production by in vitro expanded SI ILC2
(A) Flow cytometric gating strategy used to identify in vitro-expanded ILC2 from the SI of naïve mice. (B) Flow cytometric analysis of IL-5, IL-4 and IL-13 expression by Lin− CD45+ GATA-3+ ILC2 in vitro-expanded from the SI of naïve WT, Il13−/−, Il4raF709, Il4raF709 Il1rl1−/− and Il4raF709 Il4−/− mice. (C) Frequencies of IL-5+, IL-4+ and IL-13+ Lin− CD45+ GATA-3+ ILC2 isolated from the SI of the mouse groups from (B). Results represent 3 to 5 mice per group derived from 2 independent experiments. *P < .05, **P < .01, ***P < .001 and ****P < 0.0001 by one-way ANOVA with post-test analysis.
Figure 4
Figure 4. Oral allergic sensitization and anaphylaxis are obligately dependent on IL-4 production by ILC2
(A) Change in core body temperature after peanut oral challenge of peanut-sensitized Il4raF709 and Il4raF709 Il1rl1−/− mice reconstituted with either WT or Il-4−/− ILC2. (B) Peanut-specific IgE concentration and MMCP-1 release after anaphylaxis. (C – D) Frequencies and absolute numbers of ILC2 (Lin− Thy1.2+ GATA-3+) and ILC3 (Lin− Thy1.2+ RORγt+) in the MLN (C) and SI (D) from the mouse group in (A). Results represent data of minimum 7 to 18 mice per group derived from two independent experiments. *P < .05, **P < .01, ***P < .001, ****P < .0001 by two-way and one-way ANOVA with post-tests analysis.
Figure 5
Figure 5. Defective allergen-specific Treg cell induction is associated with ILC2 expansion
(A) Flow cytometric analysis of iTreg cells (CD4+ Foxp3EGFP+) in the MLN and SI of OVA-fed WT DO11.10+ Rag2−/− Foxp3EGFP and DO11.10+ Rag2−/− Il4raF709 Foxp3EGFP mice. (B) Frequencies and absolute numbers of CD4+ Foxp3+ iTreg cells in the MLN and SI of the mouse groups from (A). (C – D) Frequencies and absolute numbers of ILC2 (Lin− Thy1.2+ GATA-3+) and ILC3 (Lin− Thy1.2+ RORγt+) in the MLN (C) and SI (D) from the mouse groups in (A). Results represent data from 4 to 5 mice per group derived from two independent experiments. *P < .05, **P < 0.01, unpaired two-tailed Student’s t test.
Figure 6
Figure 6. Treg cell inhibit ILC2 expansion and their Th2 cytokine production
(A) Flow cytometric analysis of ILC2 (Lin− Thy1.2+ GATA-3+) and ILC3 (Lin− Thy1.2+ RORγt+) in the SI and MLN of Foxp3EGFP and Foxp3EGFP/DTR+ mice following DT administration. (B – C) Frequencies and absolute numbers of Lin− Thy1.2+ GATA-3+ ILC2 (B) and Lin− Thy1.2+ RORγt+ ILC3 (C) in the MLN and SI from the mouse groups in (A). (D – E) IL-4, IL-13 and IL-5 production by an increased gradient of cell-sorted WT ILC2 (D) or Il4raF709 ILC2 (E) cultured in the presence of either WT or Il4raF709 iTreg cells. (F) IL-4, IL-13 and IL-5 production by WT ILC2 cells co-cultured in the presence of Il4raF709 or IL4raF709 Il1rl1−/− iTreg cells. Results represent data from 5 to 7 mice per group. *P < .05, **P < .01, and ***P < .001 by one-way ANOVA with post-tests analysis or unpaired two-tailed Student’s t test.
Figure 7
Figure 7. IL-4 secretion by ILC2 promotes food allergy by decreasing allergen-specific Treg cell induction
(A) TGF-β induction of iTreg cells from naïve CD4+ WT or Il4raF709 T cells co-cultured with cell-sorted WT or Il4raF709 ILC2. (B) Flow cytometric analysis of the effect of ILC2 and anti(α)-IL-4 treatment on TGF-β-driven in vitro iTreg cell induction. (C) Frequency of TGF-β-induced iTreg cells co-cultured in the presence of WT ILC2 without or with αIL4. (D) Frequency of TGFβ-induced iTreg cells co-cultured with either WT ILC2, Il4−/− ILC2 or recombinant IL-4. (E) IgE-mediated mast cell degranulation following co-culture with ILC2, ILC2 supernatants or IL-4 in the presence or absence of αIL-4. (F) IgE-mediated degranulation of mast co-cultured with WT ILC2 or Il4−/− ILC2. (G) Flow cytometric analysis of peanut extract-induced in vitro proliferation of CD4+ Foxp3+ Treg cells and CD4+ Foxp3− T conventional cells isolated from the MLN of peanut-sensitized Il4raF709 and Il4raF709 Il1rl1−/− mice reconstituted with either WT or Il-4−/− ILC2 (F) Frequency of peanut-specific CD4+ Foxp3+ Treg cells and CD4+ T cells producing IL-4. Results represent data of 5 mice per group (A to F) and 7 to 18 mice (G and H) derived from two independent experiments. *P < .05, **P < .01, ***P < .001, ****P < .0001 by two-way and one-way ANOVA with post-tests analysis.
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