Immune sensing of food allergens promotes aversive behaviour

Abstract

In addition to its canonical function in protecting from pathogens, the immune system can also promote behavioural alterations ^1â€"3 . The scope and mechanisms of behavioural modifications by the immune system are not yet well understood. Using a mouse food allergy model, here we show that allergic sensitization drives antigen-specific behavioural aversion. Allergen ingestion activates brain areas involved in the response to aversive stimuli, including the nucleus of tractus solitarius, parabrachial nucleus, and central amygdala. Food aversion requires IgE antibodies and mast cells but precedes the development of gut allergic inflammation. The ability of allergen-specific IgE and mast cells to promote aversion requires leukotrienes and growth and differentiation factor 15 (GDF15). In addition to allergen-induced aversion, we find that lipopolysaccharide-induced inflammation also resulted in IgE-dependent aversive behaviour. These findings thus point to antigen-specific behavioural modifications that likely evolved to promote niche selection to avoid unfavourable environments.

Extended Data Fig. 1 |. Characterization of immunological aversion.

a, Cumulative licks of water solution from right- or left-positioned bottles from control (left) and OVA/alum sensitized mice (right). b, Percentage preference of control and OVA/alum sensitized mice to the right-side water bottle. c, Solution intake of water and varying concentrations of OVA in control BALB/c (left) and C57BL/6 (right) on day 1 of the two-bottle preference test. d, Cumulative licks to different concentrations of OVA from mice sensitized with OVA/alum. e, Preference to the bottle containing OVA in OVA/alum sensitized mice. f, Preference to 1% OVA solution by control and OVA/cholera toxin-sensitized mice. h – i, Total number of cFos positive cells in the lateral hypothalamus (LH) and paraventricular nucleus of the hypothalamus (PVN) of OVA/alum sensitized BALB/c mice. Graphs show mean ± s.e.m. Representative of at least 2 experiments.

Extended Data Fig. 2 |. Role of IgE signalling in allergic and non-allergic aversion.

a, Drinking preference of C57BL/6 WT and FcεRI KO to OVA by solution intake of different concentrations of OVA. b, Total serum IgE in WT and FcεRI KO mice sensitized with OVA/alum and orally challenged five times with OVA. c, Solution intake of OVA was measured for one week in BALB/c IL-4Ra KO (left) or Gata1Δ (right) mice orally sensitized with OVA/CT. d, Rectal temperature after systemic OVA challenge in OVA/CT orally sensitized mice. e, Total levels of serum IgE after OVA/CT sensitization and five oral challenges with OVA. f, Two-bottle preference test in C57BL/6 WT (left) or IgE KO (right) mice sensitized with OVA and LPS. g–h, Preference to OVA (g) and total serum IgE (h) in OVA/LPS sensitized WT and IgE KO mice. i, Cumulative licks during the two-bottle preference test with water and a solution with the bitter compound denatonium benzoate (DB) in C57BL/6 IgE KO mice sensitized with OVA/alum. Graphs show mean ± s.e.m. Representative of at least 2 experiments.

Extended Data Fig. 3 |. IgE-dependent and independent roles in gut allergic inflammation.

a, Experimental protocol for the development of gut allergic inflammation and systemic anaphylaxis. b, Rectal temperature over time (left) and maximum temperature variation (right) after systemic OVA challenge on day 14 in OVA/alum sensitized BALB/c WT and littermate IgE KO mice. c, Serum specific IgE (left) and IgG1 (right) antibodies in sensitized BALB/c WT and IgE KO mice after five oral challenges with OVA, on day 35. d, Gastrointestinal transit time in BALB/c WT and IgE KO mice determined with red carmine assay on day 31 upon oral allergen challenge. e–f, Accumulation of mast cells and eosinophils in the small intestines of sensitized BALB/c WT and IgE KO mice on day 35. Graphs show mean ± s.e.m. Representative of at least 3 experiments.

Extended Data Fig. 4 |. Allergen-induced avoidance behaviour is independent on substance P, CGRP, and prostaglandins.

a, c, Drinking preference to OVA solution by OVA/alum sensitized BALB/c mice that were i.p. injected with the serotonin receptor 3 antagonist (ondansetron), substance P receptor NK1 antagonist (aprepitant), CGRP receptor inhibitor (BIBN4096) or vehicle solution prior to preference test. b, Preference to OVA in C57BL/6 WT or substance P KO mice orally sensitized with OVA and cholera toxin. d, OVA preference in OVA/alum sensitized BALB/c mice administered with the cyclooxygenase inhibitor indomethacin or vehicle solution in the drinking water for 5 days prior to the preference test. e, Alox5 expression levels in the epithelial (Ep) and lamina propria (LP) compartments of the small intestine from BALB/c WT or IgE KO mice sensitized with PBS/alum (control) or OVA/alum (sensitized). n=4–5 per group. f, Staining of the dorsal motor nucleus of the vagus after fluorogold injection into the small intestine of WT mice to confirm efficiency of subdiaphragmatic vagotomy. Representative g, For TRPV1+ cell depletion, BALB/c mice were sensitized with OVA/alum, as indicated. On week 3, mice were i.p. injected with resiniferatoxin (RTX) every other day for 3 days and the two-bottle preference test was assessed one week after the last RTX injection. h, Preference to OVA solution in vehicle- or RTX-treated mice. Graphs show mean ± s.e.m., Mann-Whitney test. e, Mean ± s.e.m., One-Way ANOVA with multiple comparisons. a - h, Representative of two experiments.

Extended Data Fig. 5 |. GDF15 is induced upon allergen exposure.

a, GDF15 serum levels induced by oral (left) or intravenous (right) OVA administration in sensitized BALB/c mice. b–d, Serum GDF15 induction after oral allergen challenges in OVA/alum sensitized BALB/c and C57BL/6 (b), IgE KO (c), or mast cell-depleted RMB (d) mice. e, Induction of serum GDF15 in control and sensitized mice after the administration of the 5-lipoxygenase inhibitor zileuton during oral challenges with OVA. f, Fold change in Gdf15 mRNA transcripts in the intestinal tissues of OVA/alum sensitized BALB/c mice relative to control groups. g, Gdf15 mRNA transcripts in the duodenum (left) and colon (right) in sensitized mice. Graphs show mean ± s.e.m. One-Way ANOVA with multiple comparisons.

Extended Data Fig. 6 |. GDF15 originates from colonic epithelial cells.

a, Fcεr1a (green), EPCAM (grey), and GDF15 (red) transcripts across intestinal tissues in OVA/alum BALB/c sensitized and control mice by RNAscope. b-c, Analysis of intestinal distribution of FcεR1 expressing cells (b) and GDF15 expressing cells (c) from control and allergic sensitized WT or IgE KO mice. Quantification was performed after RNAscope technique. d, Colocalization analysis of GDF15 expressing colonic cells and cells expressing the epithelial cell marker, EPCAM. Graphs show mean ± s.e.m.

Extended Data Fig. 7 |. GDF15 blockade reduces allergen aversion

a, BALB/c mice were sensitized with OVA/alum and treated with a mouse GDF15 blocking antibody or isotype control 5 h prior to two bottle preference testing. b, Total IgE, OVA-specific IgE, and OVA-specific IgG1 antibodies after GDF15 antibody treatment. c, Cumulative licks during the two-bottle preference test of isotype control (left) or GDF15 blocking antibody-treated (right) mice. d, OVA preference of OVA/alum sensitized mice that received either GDF15 blocking antibody or isotype control 5 h prior to the behavioural assay. Graphs show mean ± s.e.m., Mann-Whitney test. e, Mean ± s.e.m., One-Way ANOVA with multiple comparisons.

Fig. 1 |. Allergic sensitization induces specific and long-lasting aversion to food allergen.

a, Schematic protocol for allergic sensitization and behavioural assay. b, Cumulative licks from mice sensitized with PBS/alum or OVA/alum. Preference test consisting of one water bottle and one 1% OVA bottle on day 1. c, Preference to OVA solution. d, Preference to OVA after 24 or 48 weeks of sensitization with alum or OVA/alum. e, Preference to bovine serum albumin (BSA). f, Preference to OVA by OVA/alum sensitized TRPM5 WT or KO mice. g, Schematic protocol of allergic sensitization and oral challenge. Mice were orally challenged with OVA after sensitization and 3 sham gavages with water. Controls received all but OVA during sensitization. h, Immunofluorescence images of the nucleus tractus solitarius (NTS, top), lateral external parabrachial nucleus (lePBN, middle), and central amygdala (CeA, bottom) from control (n=5) or OVA/alum sensitized (n=4) BALB/c mice using anti-cFos antibody, 90 min after one OVA challenge. Scale bars: 100 μm. i, Number of cFos+ neurons in the NTS, lePBN, and CeA of BALB/c control (n=5) or OVA/alum (n=4) sensitized mice. Graphs show mean ± s.e.m., Mann-Whitney test. Representative of at least 2 experiments.

Fig. 2 |. Allergic aversion requires IgE and mast cells.

a, Total (left panel) and OVA-specific (right) levels of serum IgE on day 14 after allergic sensitization in BALB/c mice. b, Cumulative licks to water and OVA solutions during the two-bottle preference test in OVA/alum sensitized BALB/c IgE KO (right) and WT littermate controls (left). c, Drinking preference to OVA bottles in controls and allergic sensitized BALB/c WT or IgE KO mice. d, Cumulative licks to water and OVA bottles in OVA/alum sensitized C57BL/6 WT or FcεRI chimeras. C57BL/6 WT or FcεRI KO bone marrow hematopoietic cells were transplanted into irradiated WT recipients. e–f, Drinking preference to OVA bottles (e) and OVA-specific IgE serum levels (f) in allergic sensitized C57BL/6 WT or FcεRI chimeras. g, Schematic protocol of FcεRI+ cell depletion with diphtheria toxin (DT) in RMB BALB/c mice. h, Cumulative licks to water and OVA bottles in allergic sensitized and DT-injected RMB WT (left) and RMB mutants (right). i–j, Preference to OVA solution (i) and OVA-specific IgE (j) in DT-injected RMB WT and mutants. Graphs show mean ± s.e.m, Mann-Whitney test. Representative of at least 2 experiments.

Fig. 3 |. Avoidance behaviour to food allergen requires cysteinyl leukotrienes.

a, Drinking preference to OVA solution by OVA/alum or PBS/alum sensitized mice. Groups were administered with antagonists for H1 and H2 histamine receptors (loratadine and famotidine), a serotonin synthesis inhibitor (parachlorophenylalanine), or vehicle solution before the two-bottle preference test. n=5–11 per group. b, Preference to OVA in OVA/alum or PBS/alum sensitized mice. Zileuton was used as a 5-lipoxygenase inhibitor 1 h prior to the preference test. n=5–14, pooled from 2 experiments. c, Alox5 expression across the GI tract of control and OVA/alum sensitized mice after allergen oral challenges. d, Alox5 expression in the duodenum of OVA sensitized WT, IgE-deficient, or mast cell-depleted mice. e, Comparison of gene expression in BALB/c intestinal immune cells from scRNA-seq data of control mice. Cluster 26 represents mast cells whereas cluster 44 refers to basophils. f, Schematic protocol for subdiaphragmatic vagotomy in OVA/alum sensitized mice. g, OVA preference after vagotomy. Graphs show mean ± s.e.m. a, b, g, Mann-Whitney test. c, d, One-Way ANOVA with multiple comparisons. a, c, d, g, Representative of at least two experiments.

Fig. 4 |. GDF15 drives allergen-induced aversion.

a, Serum levels of GDF15 in sensitized BALB/c mice after oral allergen challenges. b, Serum GDF15 in BALB/c mice after 6 oral challenges with OVA. c, Expression of GDF15 mRNA by RNAscope in the duodenum and colon of allergen sensitized and challenged WT or littermates IgE KO mice. d, OVA preference 1 h after administration of recombinant mGDF15 in WT and mast cell-depleted (MCØ) RMB mice. e, Cumulative licks on OVA bottle during preference test in OVA/alum sensitized WT mice 5 h after injection with blocking GDF15 antibody or isotype control antibody. f, Sensitized WT mice were injected with anti-GDF15 antibody, and the OVA preference was quantified 5 h later. g, Working hypothesis: allergen is sensed in the gastrointestinal mucosa of sensitized animals through allergen-specific IgE antibodies and FcεRI receptors that are highly expressed by tissue-resident mast cells. Allergen sensing by intestinal mast cells promote the release of cysteinyl leukotrienes, which mediates the secretion of GDF15 by epithelial cells in the large intestine. Systemic GDF15 likely acts directly in the brainstem, activating neurons in the nucleus of tractus solitarius (NTS) and area postrema to induce aversive circuits through the parabrachial nucleus (lePBN) and central amygdala (CeA). This regulated organismal response allows for rapid identification of allergens and the development of avoidance behaviour, evolved to limit the exposure to potentially noxious stimuli and protect from further damage. Graphs show mean ± s.e.m. a, b, d One-Way ANOVA with multiple comparisons. f, Mann-Whitney test.