Mast cells are haematopoietic cells residing in barrier tissues exposed to internal and external environments6. Mast cells are best known for their roles in immunoglobulin E (IgE)-mediated allergies, which affect up to 40% of the world’s population7. Type 2 immune responses are mounted in the context of barrier disruption and entry of infectious agents, including parasites, or tissue-damaging or innocuous protein antigens, collectively termed allergens or toxins. Type 2 immune responses, notably involving interleukin (IL)-4, drive immunoglobulin class switch recombination to antigen-specific IgE, which is bound to the mast-cell-expressed high-affinity IgE receptor (FcεRI). On reexposure to the antigen, mast cells release mediators, including proteases, histamine, serotonin and leukotrienes, which can contribute to allergic pathology. The role of mast cells in IgE-mediated allergies is often viewed as a consequence of an overreactive response using immune pathways originally directed against parasites8. In contrast to this view, Margie Profet1 proposed the hypothesis that acute allergic reactions, for instance, sneezing, coughing, vomiting and diarrhoea, may serve to rapidly expel toxins and allergens. Moreover, these symptoms would help with recognition of the source, enabling future allergen and toxin avoidance1. Although evidence for antigen-avoidance behaviour has been reported4,5, and this concept1 has been broadened2,3, no ‘immunology of avoidance’ has entered the field. Moreover, the cellular and molecular underpinnings, notably the role of mast cells, in this behavioural adaptation are poorly understood.

Here, we show that mast cells and IgE are key in promoting protein-avoidance behaviour. Mucosal mast cells lining the stomach and small intestine rapidly respond to ingested antigens. Mice harbouring mast cells but not mast-cell-deficient mice subsequently avoid antigen uptake when given a free choice of water with or without antigen under unperturbed behavioural conditions. Our findings indicate an important protective role for mast cells to signal avoidance behaviour, which, when heeded, prevents or reduces inflammation driven by repeated confrontations between the immune system and innocuous substances. The marked conservation of mast cells in animal evolution, even before the advent of IgE, indicates that immunity of avoidance may be a fundamental mode of immune defence.

Mast cells are essential for antigen-avoidance behaviour

We adapted the drink avoidance test from Cara et al.4 and made use of genetically mast-cell-deficient mice9. We induced a systemic immune response to a model protein antigen, ovalbumin (OVA), by immunizing wild-type BALB/c Cpa3+/+ and mast-cell-deficient BALB/c Cpa3Cre/+ mice9 by intraperitoneal injection on days 0 and 14 with OVA in complex with aluminium hydroxide (alum) as an adjuvant (OVA-alum) (Fig. 1a). In BALB/c mice, which are T helper type 2 prone, this induces robust OVA-specific IgE antibody responses (Fig. 1f). Control animals received alum only. Beginning on day 20, mice were subjected to the avoidance test, which is based on the preference of mice for egg white water (8% sucrose plus 20% egg white as OVA source in water) over plain water.

Fig. 1: Mast cells are essential for antigen-avoidance behaviour in type 2 immunized mice.
figure 1

a,b, Type 2 immunization scheme, experimental timeline for avoidance test (a) and IntelliCage setup with bottle positioning (b). Purple symbols indicate water, yellow symbols indicate egg white water with sucrose. c, Egg white water preferences of alum-immunized and OVA-alum-immunized BALB/c Cpa3+/+ and Cpa3Cre/+ mice displayed as percentage of egg white water intake over total water intake (colour scale indicates percentages) during the course of experiments 1 and 2. Each row corresponds to an individual mouse. d, Egg white water preference displayed as number of egg white water licks over total number of water licks versus time. Data are presented as mean ± s.e.m. e, Egg white water preference displayed as the fraction of egg white water intake over total water intake as an average per day for the duration of the experiment. f,g, Serum amounts of anti-OVA IgE (f) and anti-OVA IgG1 (g) measured at the end of the IntelliCage experiments. hj, Absolute numbers of stomach mast cells (h), frequencies of small intestine intraepithelial mast cells (i) and small intestine lamina propria mast cells (j) among total live cells, measured at the end of the IntelliCage experiments. Bars represent mean values, and each dot corresponds to a single mouse. In cj, Cpa3+/+ alum (n = 13 mice for ch, n = 12 for i and n = 13 for j); Cpa3+/+ OVA-alum (n = 16 mice for ch, n = 15 for i and n = 16 for j); Cpa3Cre/+ alum (n = 9 mice for ch, n = 7 for i and n = 9 for j); Cpa3Cre/+ OVA-alum (n = 13 mice for ch, n = 11 for i and n = 13 for j). Statistical analysis was performed using one-way analysis of variance with Tukey multiple-comparison test (ej). Exact P values are shown. i.p., intraperitoneal.

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As behavioural assays are sensitive to environmental influences and the social wellbeing of mice, we housed large cohorts of mice (13–16 mice per cage), composed of mixed experimental groups, in IntelliCages (Fig. 1b). This system allows for the uninterrupted assessment of unbiased natural behaviour (Methods). The cages were equipped with eight drinking bottles, of which four contained egg white water, and four contained plain water (Fig. 1b). Throughout the experiments, mice had the free choice of egg white water and normal water. Individual drinking preferences were continuously recorded. Non-immunized mice (alum), whether they had mast cells (Cpa3+/+) or not (Cpa3Cre/+), showed a strong preference for the egg white water during the 12 days of measurements (Fig. 1c–e). By contrast, between days 1 and 5, immunized Cpa3+/+ mice began to avoid the egg white water and afterwards drank almost exclusively normal water. Hence, in support of previous data4, Cpa3+/+ mice choose egg white water when non-immunized but normal water when immunized. Avoiding mice showed normal locomotion (number of cage corner visits; not shown) and showed no signs of disease. In marked contrast to Cpa3+/+ mice, mast-cell-deficient Cpa3Cre/+ mice failed to avoid the egg white water even when immunized (Fig. 1c–e), demonstrating an exclusive function of mast cells, that is, one that cannot be compensated by other immune or non-immune cells, for antigen-avoidance behaviour.

In summary, non-immunized mice preferred egg white water over water, whereas immunized mice shunned egg white water, and, notably, this antigen-avoidance behaviour was mast cell dependent. In addition, we observed higher egg white water preference among Cpa3Cre/+ alum versus Cpa3Cre/+ OVA-alum mice, indicating that a minor component of the OVA-avoidance response may be immunization dependent and mast cell independent (Fig. 1d).

Mast cells have been linked to anxiety-like behaviour10 that may interfere with the behavioural analysis of Cpa3Cre/+ mutant mice. To control for such potential deficits, we assessed anxiety-related and general behaviour in the elevated plus maze (Extended Data Fig. 1a–d), open field test (Extended Data Fig. 1e–i), light–dark test (Extended Data Fig. 1j–m) and recorded behaviour for 24 h in a home cage monitoring system (Extended Data Fig. 1n–s). None of these assays distinguished mast-cell-deficient mice from their wild-type littermates, indicating that the Cpa3Cre/+ mice had no behavioural deficits measurable by these assays that could have confounded the drink avoidance experiments.

The absence of OVA avoidance in immunized mast-cell-deficient Cpa3Cre/+ mice was not due to diminished OVA-specific IgE and IgG1 antibody titres compared with wild-type littermates (Fig. 1f,g). Development of aversion was associated with a pronounced accumulation of mast cells in the stomachs and small intestines of wild-type mice (Fig. 1h–j). The strongest increases were found for small intestine intraepithelial mast cells, whereas the increase in number of small intestine lamina propria mast cells was less pronounced (Fig. 1i,j). As expected, in Cpa3Cre/+ mice, numbers of mast cells in these tissues were negligible (Fig. 1h–j).

In addition to the absence of mast cells, basophil numbers are reduced to about 40% of normal in Cpa3Cre/+ mice9. To address the role of basophils, we analysed basophil-deficient Mcpt8-Cre mice (Extended Data Fig. 2). Mice were offered two bottles, one with egg white water  and one with plain water (two-bottle test). Avoidance of egg white water was unimpaired in Mcpt8-Cre mice (Extended Data Fig. 2a,b), which remained basophil deficient after immunization (Extended Data Fig. 2c,d). Except for reduced numbers of stomach mast cells, all parameters (mast cells in small intestine, anti-OVA IgG1 and IgE) were indistinguishable between basophil-deficient and wild-type mice (Extended Data Fig. 2e–h). Hence, basophils are not involved in the avoidance behaviour.

Role of IgE in antigen-avoidance behaviour

Mast cells can be activated not only by antigen and IgE through the high-affinity IgE receptor (FcεRI) but also by non-IgE stimuli11. We tested the role of IgE in antigen-avoidance behaviour. BALB/c wild-type (Igh-7+/+) and IgE-deficient (Igh-7−/−)11 mice were immunized (Fig. 1a) with OVA-alum or alum alone and subjected to the avoidance assay (Fig. 1b). In contrast to alum-immunized Igh-7+/+ and Igh-7−/− mice, OVA-alum-immunized Igh-7+/+ mice avoided the egg white water. Notably, IgE-deficient Igh-7−/− mice showed no avoidance (Fig. 2a–c). As expected, Igh-7−/− mice failed to generate anti-OVA IgE but produced anti-OVA IgG1 (Fig. 2d,e). Hence, IgE and mast cells are both essential for antigen-avoidance behaviour.

Fig. 2: Role of IgE in immunity of avoidance.
figure 2

ac, Egg white water preference of alum-immunized and OVA-alum-immunized BALB/c Igh-7+/+ and Igh-7−/− mice displayed as percentage of egg white water intake over total water intake (colour scale indicates percentages) during the course of the IntelliCage experiment (a). Each row corresponds to an individual mouse. The grey field indicates a mouse that had lost the transponder for 1 day, precluding measurement. b, Data from a displayed as egg white water licks over total water licks versus time. Data are presented as mean ± s.e.m. c, Data from a displayed as the fraction of egg white water intake over total water intake as an average per day for the duration of the experiment. d,e, Serum amounts of anti-OVA IgE (d) and anti-OVA IgG1 (e) at the end of the experiment. f,g, BALB/c Cpa3+/+ and Cpa3Cre/+ mice received mouse anti-OVA monoclonal IgE antibody on days 0 and 9 and were subjected to the avoidance test (IntelliCage as in Fig. 1b) starting on day 10 (Methods). f, Egg white water preference is displayed as in a. Each row represents an individual mouse. g, Egg white water preference is displayed as in c. h,i, Absolute numbers of stomach mast cells (h) and frequency of small intestine intraepithelial mast cells (i) among total live cells at the end of the experiment. j,k, CD63 expression, an indicator of mast cell activation, on stomach (j) and small intestine intraepithelial (k) mast cells from IgE-sensitized BALB/c Cpa3+/+ mice after intragastric gavage (i.g.) with OVA or control protein (BSA). l, Rectal temperatures after indicated challenges in IgE-sensitized (passive sensitization) or OVA-immunized (OVA-alum) BALB/c mice. Data are presented as mean ± s.e.m. For ae, Igh-7+/+ alum (n = 3 mice); Igh-7+/+ OVA-alum (n = 7); Igh-7−/− alum (n = 6); Igh-7−/− OVA-alum (n = 14). For fi, Cpa3+/+ (n = 7); Cpa3Cre/+ (n = 6). For jl, Cpa3+/+ IgE/BSA (n = 6); Cpa3+/+ IgE/OVA (n = 5); Cpa3+/+ OVA-alum/OVA (n = 2). Statistical analysis was performed using one-way analysis of variance with Tukey multiple-comparison test (ce) and two-sided Student’s t tests (gk). Exact P values are shown. MFI, mean fluorescence intensity.

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To test whether IgE is sufficient in the absence of immunization, we injected monoclonal anti-OVA IgE into BALB/c mice. This led to partial sensitization of wild-type but not mast-cell-deficient mice (Fig. 2f,g). Four of seven wild-type mice showed variable degrees of avoidance (Fig. 2f,g). Of note, after transfer of IgE, mast cell levels in stomach and small intestine were not elevated (Fig. 2h,i for passive sensitization; Fig. 1h,i for active immunization). In line with the partial avoidance response (Fig. 2f,g), only half of the mice showed activation of stomach mast cells on OVA contact (Fig. 2j), whereas none of them showed activation of small-intestinal epithelial mast cells (Fig. 2k). Moreover, active immunization but not IgE transfer sensitized BALB/c mice for anaphylaxis (based on temperature drop) on intragastric OVA application (Fig. 2l). Hence, monoclonal IgE transfer can partially induce avoidance behaviour; however, immunization may be required to attain full mast cell activation and avoidance.

Th2 cytokines promote antigen-avoidance behaviour

We analysed a Th1-biased strain with (C57BL/6 Cpa3+/+) or without (C57BL/6 Cpa3Cre/+)9 mast cells with respect to its ability to mount antigen-avoidance behaviour responses. Owing to their elevated sensitivity to sucrose12, even at only 1% (as opposed to 8% in the BALB/c experiments), all but one of the OVA-alum-immunized C57BL/6 Cpa3+/+ mice preferred egg white water (Extended Data Fig. 3a,d). However, at lower sucrose concentration (0.25%), avoidance behaviour became evident in C57BL/6 Cpa3+/+ mice (Extended Data Fig. 3b,e), as well as its mast cell dependency in the C57BL/6 Cpa3Cre/+ mice (Extended Data Fig. 3b,e). Antigen avoidance was not observed in C57BL/6 mice immunized with alum only (Extended Data Fig. 3a,b,d,e).

C57BL/6 mice were injected with a Th2-promoting and mast-cell-promoting cytokine cocktail 2 days after each round of immunization (Methods). This treatment enhanced the avoidance response; however, this response remained dependent on mast cells and OVA immunization (Extended Data Fig. 3c,f). Compared with that of BALB/c mice, the avoidance response of C57BL/6 mice remained less pronounced even under cytokine stimulation (Fig. 1c versus Extended Data Fig. 3c). Whereas OVA-specific IgE levels and stomach mast cell counts were comparable (Fig. 1f,h versus Extended Data Fig. 3g,h), levels of small intestine intraepithelial mast cells were approximately eight-fold higher in BALB/c mice (Fig. 1i) compared with cytokine-stimulated C57BL/6 mice (Extended Data Fig. 3i), indicating a mast cell compartment with potential relevance for avoidance. Although mast cells in both stomach and small intestine were activated on oral OVA administration in immunized C57BL/6 mice (Extended Data Fig. 3j–l; measurement of activation is provided in the context of Fig. 4), systemic anaphylaxis required intravenous OVA injection (Extended Data Fig. 3m). In summary, Th2 cytokines promote mast-cell-dependent avoidance behaviour in OVA-immunized, Th1-prone C57BL/6 mice. However, in contrast to the BALB/c strain (Fig. 2l), OVA-immunized C57BL/6 mice were resistant to anaphylaxis by oral antigen challenge.

Antigen avoidance prevents inflammation

We examined immunological consequences in the gastrointestinal tract, focusing on the stomach and small intestine, under avoidance and non-avoidance conditions. To this end, wild-type BALB/c mice were immunized and subjected to either the two-bottle test (‘avoidance’) or OVA gavage (‘non-avoidance’) (Methods) for up to 16 days (Extended Data Fig. 4a). Mast-cell-deficient BALB/c mice were subjected to the same protocol. As expected, wild-type mice avoided whereas mast-cell-deficient mice preferred egg white water in the two-bottle test (Extended Data Fig. 4b). Wild-type mice forced to ingest OVA by gavage developed diarrhoea, whereas all but one of the mast-cell-deficient mice remained healthy after OVA gavage (Extended Data Fig. 4c), consistent with the role of mast cells in models of food allergy13. Occurrence of diarrhoea indicated induction of inflammatory and immunological processes under non-avoidance conditions. Indeed, the percentages of neutrophils in stomach and small intestine increased, and serum (hence systemic) levels of IL-4 and IL-6 were elevated (Extended Data Fig. 4d–g).

We analysed changes in gene expression by RNA sequencing (RNA-seq) in whole lysates from stomach and small intestine taken on days 7, 11 and 16 after the beginning of the tests (Extended Data Fig. 4a). We reasoned that RNA-seq would yield comprehensive, high-resolution data on possible immune pathway activation and inflammation, comparing non-avoidance with avoidance conditions. Samples were subjected to principal component analysis (Extended Data Fig. 5a–d) based on the 500 most variable genes, corresponding to approximately 45% of variance. The results indicated the greatest differences between wild-type mice under avoidance (two-bottle test) and non-avoidance (OVA gavage) conditions (Extended Data Fig. 5a,b). This distinction was less obvious (in the small intestine) or absent (in the stomach) in mast-cell-deficient mice, which were non-avoiding in both the two-bottle test and under gavage (Extended Data Fig. 5c,d).

We characterized differential gene expression in the stomach and small intestine, comparing avoidance and non-avoidance conditions (Extended Data Fig. 5e,f). We first compared wild-type mice under avoidance (two-bottle test) and non-avoidance (gavage) conditions, to shed light on any gastrointestinal antigen-driven immunity and inflammation that is prevented by antigen-avoidance behaviour in mice with a normal immune system. For all time points and tissues, expression of genes indicative of adaptive, innate and mucosal immunity as well as chemotaxis was elevated under non-avoidance conditions (Extended Data Fig. 5e,f and Supplementary Tables 1 and 2).

To identify differentially expressed genes (DEG) characteristic of avoidance or non-avoidance, we analysed fold changes in gene expression versus P values using volcano plots (Fig. 3a–d). In this manner, we compared avoiding mice (two-bottle test, where mice still drink small amounts of the OVA solution) with mice receiving only water (Fig. 3a,c). We also compared mice receiving gavage (non-avoidance) with mice drinking only water (Fig. 3b,d). Under non-avoidance conditions, DEG composed of mast-cell-related and immunity-related genes (for definitions, see Methods) emerged in the stomach and small intestine (Fig. 3b,d) (Supplementary Table 3). No transcriptional response of this scope and magnitude was observed in avoiding mice (Fig. 3a,c). Notably, even avoiding mice showed some gene activation compared with water-only mice, including activation of mast-cell-related genes (Fig. 3a,c). This gene activation, which only became evident by comparison of water-only versus avoiding mice, may define a threshold for the amount of voluntary antigen uptake in antigen-sensitized mice (Fig. 3a versus Fig. 3b and Fig. 3c versus Fig. 3d).

Fig. 3: Antigen avoidance prevents immune activation and inflammation in gastrointestinal tissues.
figure 3

ad, Volcano plots of DEG in stomach (a,b) and small intestine (c,d) tissues comparing avoiding mice (7-day two-bottle test; stomach n = 4, intestine n = 3) with mice receiving only water (stomach n = 2, intestine n = 3) (a,c) and non-avoiding mice (7-day gavage; stomach n = 3, intestine n = 3) with mice receiving only water (stomach n = 2, intestine n = 3) (b,d). The experimental outline is shown in Extended Data Fig. 4a. Red dots represent genes associated with immunity-related GO terms (Methods and Extended Data Fig. 5e, f). Blue dots represent manually annotated mast cell genes. e, Heatmaps of log2 fold change in gene expression in stomachs and small intestines of mice under avoidance and non-avoidance conditions on day 7. Genes shown are taken from b and d, satisfying log2 fold change >3 and P ≤ 0.05. Fold changes are indicated by scale: white indicates a gene not significantly regulated. All data are from wild-type BALB/c mice. Statistical comparisons were done using the DESeq2 package (Methods). P values were adjusted for multiple comparisons using the Benjamini–Hochberg algorithm. FC, fold change; NS, not significant.

For visualization of tissue-specific signatures and annotation of genes in addition to those related to mast cells and immunity, we generated heatmaps of signature genes (Fig. 3e; for gene filtering, see Methods). We identified genes associated with tissue remodelling and antimicrobial functions, which, notably, showed differences between the stomach and small intestine (Fig. 3e). Finally, we also probed our DEG versus a published inflammatory gene expression list14. In both stomach and small intestine, the inflammatory signature was evident under non-avoidance but not avoidance conditions in wild-type mice (Extended Data Fig. 5g,h and Supplementary Table 4). Collectively, our comparison of gastrointestinal gene expression at a tissue level between mice under avoidance and non-avoidance conditions showed broad immune activation and inflammation in stomach and small intestine, nearly all of which was prevented by antigen-avoidance behaviour.

Immune activation in non-avoiding Cpa3 Cre/+ mice

Next, we asked how much of the non-avoidance induced immune-related and inflammation-related response was mast cell dependent. In analogy to the analysis in Extended Data Fig. 5e,f, we compared gene expression in BALB/c wild-type (avoiding) versus mast-cell-deficient (non-avoiding) mice in the two-bottle test (following the experimental outline in Extended Data Fig. 6a). In mast-cell-deficient mice, the induction of immune response genes was much reduced and its kinetics were altered (Extended Data Fig. 6b,c), compared with wild-type mice under non-avoidance conditions (Extended Data Fig. 5e,f). In the stomachs of mast-cell-deficient mice, enhanced immune gene expression was only detected on days 11 and 16 (Extended Data Fig. 6b). The intestines of mast-cell-deficient mice showed elevated immune gene expression only on day 7 (Extended Data Fig. 6c). Hence, the immunological and inflammatory response that is prevented by mast-cell-mediated antigen-avoidance behaviour is largely but not exclusively driven by mast cells.

Mast cell sensing of ingested antigens

To identify OVA-responsive mast cells along the route from the oral cavity to the gastrointestinal tract, we immunized Nr4a1-green fluorescent protein (Nr4a1-GFP) reporter mice. This enabled in vivo tracing of mast cells activated through FcεRI15 (Fig. 4a). Three hours after OVA-immunized Nr4a1-GFP mice had drunk 25% OVA water, we isolated cell suspensions and analysed mast cells by flow cytometry for activation. Mast cells from the oral cavity (gingiva and tongue) remained GFP-negative, whereas stomach and small-intestinal mast cells showed pronounced signalling reporter expression (Fig. 4b and Extended Data Fig. 7a). We also analysed the oesophagus and colon but detected no mast cells (oesophagus) or only minute numbers (colon), precluding further analysis (not shown). In a direct demonstration of their antigen reactivity, freshly isolated stomach mast cells from immunized but not control mice (BALB/c) showed specific increases in intracellular Ca2+ in response to OVA stimulation in vitro (Extended Data Fig. 7b,c). Collectively, these results show that oral antigen exposure rapidly activates tissue-resident stomach and small-intestinal mast cells.

Fig. 4: In situ tracing of mast cell activation and role of FLAP in antigen avoidance.
figure 4

a, Model for detection of FcεRI-activated mast cells using the Nr4a1-GFP reporter allele. In vivo OVA-responsive mast cells expressed GFP. b, (BALB/c x C57BL/6)F1 Nr4a1-GFP mice immunized with OVA-alum or alum only were given drinking water with OVA or BSA (Methods). Representative histograms (for data shown in Extended Data Fig. 7a) of GFP expression in tissue mast cells isolated ex vivo after drinking OVA- or BSA-containing water for 3 h. c, Model displaying the generation of leukotrienes and FLAP inhibition by MK-886. d,e, BALB/c wild-type mice were immunized with OVA-alum (as in Fig. 1a), and, 1 h before the IntelliCage (d), or two-bottle (e) test, mice were treated with PBS or MK-886 (Methods). Preference is displayed as the fraction of egg white water intake over total water intake for 6, 12 or 24 h. Bars represent mean values, and each dot corresponds to a single mouse. Alum vehicle (n = 5 mice), alum MK-886 (n = 5), OVA-alum vehicle (n = 8), OVA-alum MK-886 (n = 12) (d), alum vehicle (n = 14), alum MK-886 (n = 14), OVA-alum vehicle (n = 19), OVA-alum MK-886 (n = 19) (e). Statistical analysis was performed by two-sided Student’s t tests in d and e. Exact P values are shown. AA, arachidonic acid. Figure 4a created with

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Role of leukotrienes in antigen-avoidance behaviour

After mucosal antigen contact, mast cells become activated to release de novo synthesized lipid mediators and preformed mediators including biogenic amines, proteoglycans and proteases from their granules16. Lipid mediators, such as leukotrienes and prostaglandins, can activate sensory neurons17.

Leukotrienes are generated from phospholipids by cytosolic phospholipase A2 and 5-lipoxygenase (5-LO); the latter requires the 5-LO-activating protein (FLAP), which is thus essential for the generation of leukotrienes18 (Fig. 4c). We sought to specifically test the contribution of leukotrienes to OVA avoidance by pharmacological inhibition of FLAP. To this end, BALB/c wild-type mice were alum or OVA-alum immunized and treated, 1 h before the avoidance tests, with FLAP inhibitor MK-886 (ref. 19). FLAP inhibition reduced avoidance over the course of 24 h, with P values reaching significance at 6 h (Fig. 4d, Experiment 1) or 24 h (Fig. 4e, Experiment 2) compared with vehicle-treated controls. This inhibitor effect was seen in around half of the mice. These data indicate that leukotrienes may be involved at least in initial antigen-avoidance behaviour.

Next, we tested the roles in antigen avoidance of preformed mast cell mediators, including carboxypeptidase A3 (Cpa3) using Cpa3Y356L,E378A mutant mice (Extended Data Fig. 8a), mast cell protease 5 (Mcpt5) using Cpa3−/− (double deficient for Cpa3 and Mcpt5) mice (Extended Data Fig. 8b), mast cell tryptase (Mcpt6) using Mcpt6−/− mice (Extended Data Fig. 8c) and histamine using histidine decarboxylase (Hdc) Hdc−/− mice (Extended Data Fig. 8d). Immunized mice of these strains developed OVA avoidance to the same extent as wild-type mice (Extended Data Fig. 8a–d), excluding functions for Cpa3, Mcpt5, Mcpt6 and histamine in OVA-avoidance behaviour.

One of the avoidance signature genes enriched in mice force fed with antigens is Tph1 (Fig. 3b,d,e), the enzyme for serotonin (5-hydroxytryptamine; 5-HT) synthesis. A large fraction of stomach mast cells harboured 5-HT (Extended Data Fig. 8e,f). Mast-cell-derived 5-HT may signal through its ionotropic serotonin 3 receptor (5-hydroxytryptamine receptor 3; 5-HTR3), a key regulator of visceral malaise, nausea and emesis20. We tested the role of 5-HTR3 in antigen avoidance by treatment of mice with the specific inhibitor palonosetron21. Palonosetron did not significantly decrease antigen-avoidance behaviour (Extended Data Fig. 8g). In summary, of all the preformed or newly generated mast cell mediators that we tested, only leukotriene synthesis blockade inhibited antigen-avoidance behaviour.

Interrogation of neural pathways

The fast avoidance reaction of some immunized mice (29% of wild-type mice within the first day and some even immediately after their first licks) (Extended Data Fig. 9a,b) could be due to rapidly acting humoral factors in the blood circulation or direct signalling from mast cells to neurons. The small-intestinal epithelium is innervated by intrinsic primary afferent neurons residing in submucosal and myenteric plexi of the enteric nervous system or by extrinsic neurons located in the dorsal root ganglia and vagal ganglia22 (Extended Data Fig. 10a). We investigated possible mast cell signalling to intrinsic neurons by ex vivo calcium imaging of intestine segments from immunized mice expressing a genetically encoded calcium sensor in submucosal plexus (Extended Data Fig. 10b,c) and myenteric plexus (Extended Data Fig. 10d–f) neurons. Avoidance signalling through extrinsic vagal neurons was assessed by subdiaphragmatic vagotomy of immunized mice (Extended Data Fig. 10g,h). In addition, we tested the effect of resiniferatoxin (RTX)-mediated depletion of extrinsic Trpv1-expressing vagal and dorsal root ganglion sensory neurons on antigen avoidance (Extended Data Fig. 10i,j). None of these experiments revealed evidence for a neuronal route transmitting the avoidance signal (Extended Data Fig. 10b–j). As Trpv1-expressing (RTX-sensitive) neurons represent only a subset of all dorsal root ganglion neurons23, a function for RTX-insensitive dorsal root ganglion neurons cannot be ruled out.


We show here that type 2 immunized animals sense antigens rapidly by mast cells lining the stomach and small intestines. Antigen-avoidance behaviour was found to be dependent on mast cells and IgE and was amplified by Th2 cytokines. Comparison of voluntary with no antigen uptake demonstrated a threshold for antigen consumption, resulting in apparently ‘acceptable’ low-level immune activation in the gastrointestinal tract. Ingestion of antigen above this threshold, which would normally be prevented by avoidance behaviour, resulted in strong gene activation indicative of adaptive, innate and mucosal immunity, as well as chemotaxis. Such broad and sustained (for at least 16 days) elevation of expression of immune-related and inflammation-related gene programmes indicates a profound immune response to OVA, akin to food allergy. The gastrointestinal epithelium forms an important barrier controlling nutrient uptake and preventing entry of microbes across the intestinal wall. Activated mast cells may induce intestinal leakiness by secretion of proteases Mcpt1 (ref. 24) and Mcpt4 (ref. 25), and indeed the Mcpt1 and Mcpt4 genes were strongly upregulated under non-avoidance conditions (Fig. 3e). The non-avoidance signature also contained genes associated with antimicrobial function, including Sprr2a3, Itnr and Reg3g, and tissue remodelling, including Has1, Adamts4 and Apold1, which may indicate impaired barrier function. Hence, avoidance behaviour prevents antigen-driven immune activation, ensures barrier integrity and protects against allergic pathology13,26,27. The extent of immune gene induction was lower in non-avoiding mast-cell-deficient mice compared with force-fed wild-type mice, indicating that immune activation may be largely but not completely mast cell dependent.

The conservation of mast cells and IgE over millions of years in mammalian species strongly implies that these immune components provided a robust evolutionary advantage. Experimentally, when anaphylaxis was discovered in 1901 by Richet and Portier28, it was interpreted as a fatal, ‘non-protecting’ response against the toxin with which dogs were repeatedly inoculated. The immunological ‘purpose’ of allergic responses has been a matter of long-lasting debate1,2,8. Whereas mast cells and IgE contribute to allergic pathology, there are few examples for protective Th2 responses through mast cells29 and IgE29,30. Beneficial functions of mast cells and IgE are commonly invoked in parasite immunity31. However, parasite infections may be unperturbed32 or only delayed in the absence of mast cells33, and functions of mast cells and IgE can be redundant with those of other cells (for instance, eosinophils34) or antibody isotypes35,36. Given the complexities in understanding the actual roles of mast cells in immunological protection, the role for mast cells observed here in antigen-avoidance behaviour is intriguing. The non-redundant function of mast cells in the avoidance reaction may represent an evolutionary advantage of preventing recurrent immune responses to non-infectious antigens (allergens) and intoxications. Although allergens are frequently innocuous, non-toxic substances2 (see, for example, the long list of occupational allergens37) and remain non-immunogenic in the presence of intact barriers, they can elicit type 2 responses in the context of barrier defects in gut, lung or skin. Irritants such as detergents or solvents can damage the integrity of inner and outer surfaces38, and antigen-specific IgE production can be enhanced in the wake of barrier defects39. Whether a substance is both barrier damaging and immunogenic, for instance, papain40 or Derp1 (ref. 41), or whether a physical or chemical insult or an infection26 damages barriers and opens the door to immunity against innocuous substances is irrelevant to the outcome: type 2 immunity, IgE production and loading of mast cells with antigen-specific IgE (Fig. 5). For both toxins and innocuous substances, avoidance prevents repeated contact, which, as we show here for a protein antigen, results in local and systemic immune activation and inflammation.

Fig. 5: Mast cells and IgE promote immunity of avoidance.
figure 5

Model of mast-cell-mediated avoidance behaviour in the framework of type 2 immunity. Barrier damage facilitates entry of antigens (for instance, occupational and innocuous proteins such as those from flour, eggs or peanuts, as well as from pathogens), leading to a type 2 immune response. Adaptive immunity generates specific IgG and IgE antibodies towards antigen neutralization. Antigen-specific IgE binds to mast cells, which, on reencounter with antigens, signal avoidance behaviour. Inhibition of FLAP-dependent leukotrienes impairs avoidance, indicating that this mediator may contribute to the immune–brain axis. Mice avoiding antigen (heeding avoidance) are largely protected from developing gastrointestinal immune activation and inflammation, which occur when avoidance is ignored. Immunity of avoidance is dependent on the presence of mast cells and IgE (not shown). B, B cell; DC, dendritic cell. Created with

Owing to their location in barrier tissues and their close proximity to sensory neurons42, mast cells are poised to sense protein antigens and signal their presence to the central nervous system. The compounds secreted by activated mast cells include leukotrienes16. Leukotrienes can activate sensory neurons, as has been shown for LTC4, which activates spinal neuron pathways through CysLT2R to induce itch17. It is thus conceivable that leukotrienes released from mast cells following antigen sensing signal avoidance within the first 24 h via dorsal root ganglion and spinal neuron pathways. Our analysis of antigen consumption kinetics in the IntelliCage shows that avoidance frequently develops on first oral antigen contact. A function for leukotrienes at this stage would be consistent with our experiments using an inhibitor of leukotriene production43 (Fig. 4).

Aversive signals are processed in the brain by the parabrachial nucleus (PBN), which receives input from the spinal cord and brain stem44. Activation of neurons in the PBN is sufficient to drive conditioned taste avoidance after experimentally pairing a novel food (for instance, a sweetened solution) with injection of malaise-inducing cisplatin45. In this setting, cisplatin injection causes systemic release of growth and differentiation factor 15 (GDF-15) into the circulation45, which signals avoidance of the paired food to the PBN through GFRAL-positive brain stem neurons45,46,47. Florsheim et al. propose that GDF-15 is induced downstream of mast cells and leukotrienes to promote antigen avoidance43. GDF-15 blockade effectively reduced avoidance during the second but not the first drink test. In this phase, systemic release of GDF-15 and signalling through brain stem neurons may sustain avoidance.

The sensing of environmental antigens by mast cells extends immunity beyond the neutralization and destruction of pathogens to the prevention of inflammatory disease by antigen avoidance. Despite their bone marrow origin, mast cells are only distantly related to other immune lineages48,49; it is thus not surprising that they have separate, unique functional properties. The present work identifies them as sensor cells linking antigen recognition elicited by type 2 immune responses to behaviour. In this manner, at long last50, mast cells emerge with an important and non-redundant new function.



BALB/c Cpa3Cre/+ (ref. 9), C57BL/6 Cpa3Cre/+ (ref. 9), BALB/c Mcpt8-Cre51, BALB/c Cpa3−/− (ref. 52), BALB/c Cpa3Y356L,E378A (ref. 53), BALB/c Hdc−/− (ref. 54), BALB/c Mcpt6−/− (ref. 55) and Nr4a1-GFP56 mice on C57BL/6 or (BALB/c × C57BL/6)F1 background were maintained in the mouse breeding facilities of the DKFZ Heidelberg. BALB/c Igh-7−/− (ref. 11) mice were bred at Imperial College London and used at the Interdisciplinary Neurobehavioral Core, Heidelberg University, for experiments. BALB/c wild-type mice and C57BL/6 Wnt1|GCaMP3 (Wnt1-Cre;R26R-GCaMP3)57,58 mice were maintained in the mouse breeding facilities of the KU Leuven. Mice were housed with a 12-h day–night cycle in a controlled environment at 20–24 °C and 45–65% humidity. All behavioural tests were performed on adult (>7 weeks old) female and male mice. Controls for Cpa3Cre/+, Mcpt8-Cre, Cpa3Y356L,E378A, Cpa3−/− and Mcpt6−/− mice were gender-matched and age-matched wild-type littermates. Controls for Igh-7−/− and Hdc−/− mice were gender-matched and age-matched wild-type mice housed in the same animal room. All animal experiments were performed in accordance with institutional and governmental regulations. Experiments in Heidelberg were approved by the Regierungspräsidium Karlsruhe, Germany. Experiments in Leuven were approved by the Animal Care and Animal Experiments Committee of the KU Leuven, Leuven, Belgium.


Mice were actively immunized by intraperitoneal injection with 25 μg OVA (Sigma-Aldrich) complexed with 2 mg Al(OH)3 (alum) (InvivoGen) on days 0 and 14, and avoidance tests started on day 20. For Th2 cytokine treatment, mice were injected intraperitoneally with a cocktail composed of 2 μg IL-3 (Peprotech), 12 μg anti-mouse IL-3 (MP2-8F8, BioLegend), 2 μg IL-4 (Peprotech), 12 μg anti-mouse IL-4 (11B11, BioLegend) and 0.4 μg IL-9 (Peprotech) on days 2 and 16. Mixing IL-3 with the anti-IL-3 antibody MP2-8F8 and IL-4 with the anti-IL-4 antibody 11B11 generates cytokine–antibody complexes that show increased activity in vivo59, which we exploited here to increase the magnitude and duration of cytokine effects in vivo. No such effects have been described for IL-9; hence no anti-IL-9 antibody was used. For passive sensitization, mice were injected intraperitoneally on day 0 and intravenously on day 9 with 10 μg anti-OVA IgE monoclonal antibody (E-C1, Chondrex), and intragastric challenge (anaphylaxis) or avoidance test started on day 10.


Keeping mice in IntelliCages prevents stress induced by handling and thus enables observation of natural behaviour60. For systemic immunization, mice received two injections of OVA-alum on days 0 and 14 as described above. On day 17, mice received a subcutaneous implant of a unique radio-frequency identification (RFID) transponder into the nape under isoflurane anaesthesia and were placed in an IntelliCage apparatus (TSE Systems) in groups of 13–16 animals. Mice were kept on a 12-h light–dark cycle with ad libidum access to chow and water. After acclimatization (1–3 days), the water in four of eight bottles was exchanged for 20% (v/v) egg white water containing either 0.25% (w/v), 1% (w/v) (C57BL/6) or 8% (w/v) (BALB/c) sucrose. Drinking behaviour was analysed for up to 14 days. Data were collected using the IntelliCage Plus software (NewBehavior AG).

For passive sensitization, mice received two injections of monoclonal anti-OVA-IgE on days 0 and 9 as described above. On day 11, mice received a subcutaneous implant of a unique RFID transponder into the nape under isoflurane anaesthesia and were placed in the IntelliCage apparatus. Mice were kept on a 12-h light–dark cycle with ad libidum access to chow and water. After acclimatization, the water in four of eight bottles was exchanged for 20% (v/v) egg white water containing 8% (w/v) sucrose. Drinking behaviour was analysed for up to 14 days. Data were collected using the IntelliCage Plus software (NewBehavior AG).

Egg white preparation

Egg white (20% v/v) was prepared as follows: chicken egg white was separated from its yolk, diluted in water and strained through filter paper (grade 3 hw; 65 g m−2; Ahlstrom Munksjö). The 20% egg white solutions used for experiments contained on average 0.78 ± 0.17 endotoxin units per millilitre, which is substantially lower than that of standard mouse chow61.

Two-bottle test

One week after the second immunization, BALB/c Cpa3+/+ and BALB/c Cpa3Cre/+ or knockout mice and their corresponding littermates were individually housed. Cages were equipped with two identical bottles, one containing water and the other containing 20% egg white water with 8% sucrose. According to the results of sodium dodecyl sulfate polyacrylamide gel electrophoresis, the 20% egg white water solution contained approximately 40 mg ml−1 OVA (not shown). Every 24 h, bottles were weighed, and the positions of the bottles were changed to control for a side preference in drinking behaviour. The avoidance tests were run for up to 7 days.

Multiple OVA gavage treatments

Mice received 50 mg OVA by intragastric gavage every 2–3 days (for 7 days treatment, 4× gavage; for 11 days, 6× gavage; for 16 days, 8× gavage). Mice were inspected for diarrhoea 1 h after each gavage. By comparison to this OVA gavage, voluntary consumption by mast-cell-deficient BALB/c Cpa3Cre/+ mice in IntelliCage experiments (Fig. 1) was on average 586 mg OVA per day. Hence, the amount of antigen given by gavage was not higher than the amount of antigen consumed voluntarily by mast-cell-deficient mice.

Pharmacological inhibition of FLAP and 5-HTR3

Mice were immunized with OVA-alum as described above. On day 20, mice were deprived of drinking water overnight. Palonosetron (Sigma-Aldrich) was injected intraperitoneally at a dose of 0.5 mg kg−1 12 h before the avoidance test. MK-886 (Abcam) was administered by intragastric gavage at a dose of 10 mg kg−1 1 h before the avoidance test. As repeated dosing with MK-886 can alter mouse behaviour62, we only gave the mice a single dose, followed by a 1-day observation. IntelliCage (MK-886) and two-bottle tests (MK-886; palonosetron) were run for 24 h.

Analysis of mast cell activation in Nr4a1-GFP mice

Mice were immunized with OVA-alum as described above. On day 20, Nr4a1-GFP mice were housed individually. After a 12-h period of water deprivation, cages were equipped with a bottle containing 25% OVA (Sigma-Aldrich) in water. Control mice were given a bottle containing 25% bovine serum albumin (BSA, Roth) in water. After 3 h, the bottles were weighed to check for consumption from the bottles, and mice were euthanized for further analysis. Activation of purified mast cells was monitored by flow cytometry for GFP expression.

For analysis of anaphylaxis in Nr4a1-GFP mice, mice were immunized with OVA-alum as described above. On day 21, mice received intragastric gavage with 50 mg OVA (Sigma) or 50 mg BSA (Sigma), and their body temperature was monitored using a rectal thermometer. After 3 h, mice were euthanized for further analysis. Activation of purified mast cells was monitored as described above.

Pharmacological ablation of Trpv1-positive sensory neurons

Mice were immunized with OVA-alum as described above. Between the OVA immunizations, starting on day 10, resiniferatoxin (Cayman Chemicals) was injected subcutaneously into the flank in three escalating doses (30, 70 and 100 μg kg−1) on consecutive days. Control mice were treated with vehicle (dimethyl sulfoxide in phosphate-buffered saline (PBS)). On day 20, Trpv1 denervation was confirmed by prolonged withdrawal latency of mice to noxious heat (52 °C) applied to the tail (tail flick test; data not shown). Beginning on the next day, OVA avoidance was analysed by the two-bottle test.

Tissue digestion

Gingival single-cell suspensions were prepared as previously described63. In brief, the palate and mandible were isolated, and tissues were digested for 1 h at 37 °C in RPMI supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich), 0.15 μg DNase I and 3.2 mg ml−1 collagenase IV (all enzymes from Sigma-Aldrich). Then, 0.5 M EDTA (Roth) was added during the last 5 min, and supernatant was filtered through a 70-μm cell strainer (ThermoFisher). Undigested gingiva tissue was peeled from the palate and mandible and mashed through the same filter to yield the gingiva cell suspension.

Tongue single-cell suspensions were prepared by finely mincing the tongue and digesting the tissue for three rounds of 15 min at 37 °C in RPMI supplemented with 0.1 mg ml−1 Liberase (Sigma-Aldrich) and 2.5 μg ml−1 DNase I (Sigma-Aldrich). After each round of digestion, the cell suspensions were filtered through a 70-μm cell strainer (ThermoFisher), and new enzyme solution was added to the tissue. All fractions were combined to yield the tongue single-cell suspension.

For isolation of stomach intraepithelial leucocytes, the stomach was cut open and food remnants were removed. Stomachs were incubated for 15 min at 37 °C in HBSS supplemented with 20 mM EDTA (Roth) to release epithelial layers from the connective tissue. The cell suspension was applied to a spin column (ThermoFisher) packed with 100-μm zirconia beads (Roth). After centrifugation, the flowthrough was collected, yielding an intraepithelial cell suspension containing mucosal stomach mast cells.

For preparation of small intestine cell suspensions, small intestines were cut open and food remnants were removed. Intestines were incubated for 15 min at 37 °C in HBSS supplemented with 2% FCS (Sigma-Aldrich), 5 mM EDTA (Roth), 1 mM DTT (Merck) and 10 mM HEPES (Life Technologies) to release epithelial layers from the connective tissue27. The cells in the soluble fraction (containing intraepithelial mast cells) were filtered through a 70-μm cell strainer (ThermoFisher). The remaining intestine tissue was washed in PBS and transferred into RPMI supplemented with 2% FCS (Sigma-Aldrich), 20 mM HEPES (Life Technologies), 0.2 mg ml−1 collagenase IV (Sigma-Aldrich), 0.5 mg ml−1 hyaluronidase I (Sigma-Aldrich) and 0.1 mg ml−1 DNase I (Sigma-Aldrich). Digestion was carried out for 30 min at 37 °C, and digested tissue was filtered through a 100-μm cell strainer (ThermoFisher), yielding the lamina propria fraction (containing lamina propria mast cells).

Blood was drawn by cardiac puncture, followed by red blood cell lysis according to the manufacturer’s protocol (RBC Lysis Buffer, BioLegend).

Flow cytometry

Single-cell suspensions were centrifuged and incubated for 15 min with 200 μg ml1 mouse IgG (Jackson ImmunoResearch Laboratories) to block Fcγ receptors. After washing with PBS supplemented with 5% FCS (Sigma-Aldrich), cells were stained with fluorochrome-coupled antibodies (see list in the Antibodies section) for 20 min on ice and protected from light. Cells were washed and incubated with 100 nM SytoxBlue (Life Technologies) for dead cell exclusion. For absolute quantitation of cells, a defined number of 123 count eBeads (Life Technologies) were added to the samples before analysis with a BD LSRFortessa (Becton Dickinson). Data were analysed using FlowJo software (Treestar), using the gating strategies shown in Supplementary Fig. 1. Mast cells in the tongue and gingiva were gated as live CD45+MHCIICD11bCD117+FcεRI+ cells. Stomach mast cells were gated as live CD45+CD117+FcεRI+/IgE+ cells. Intestinal mast cells were gated as live CD45+CD3CD11bCD19Gr-1Ter119CD117+FcεRI+ cells. Neutrophils were gated as live CD45+CD11b+Siglec-F Gr-1+/Ly6G+ cells. Basophils were identified as live CD45+CD90.2CD11cGr-1Siglec-FMHCIIB220CD49b+IgE+ cells. For reagents, see list in the Antibodies section.

Intracellular Ca2+ measurement of mast cells

For analysis of Ca2+ flux in stomach mast cells, stomach intraepithelial leucocytes were spun down and resuspended in calcium imaging buffer (125 mM NaCl, 3 mM KCl, 2.5 mM CaCl2, 0.6 mM MgCl2, 10 mM HEPES, 20 mM glucose, 1.2 mM NaHCO3 and 20 mM sucrose, brought to pH 7.4 with NaOH) supplemented with 0.1% BSA (Roth), 2.5 mM probenecid (Biotinum), 0.01% Pluronic-F127 (Sigma-Aldrich) and 200 μg ml1 mouse IgG (Jackson ImmunoResearch Laboratories) to block Fcγ receptors. After washing with calcium imaging buffer, cells were stained with 4 μM Fluo-4 (Thermo Fisher Scientific) and CD45 BV421, CD117 PE and FcεRI APC antibodies for 30 min at room temperature in the dark. Cells were then washed with calcium imaging buffer supplemented with 0.1% BSA (Roth) and 100 nM SytoxBlue (Life Technologies). Cells were kept at 37 °C during measurements and analysed on a BD LSRFortessa (Becton Dickinson). After 30 s of baseline measurements, OVA (Sigma-Aldrich) was added to a final concentration of 1.25 mg ml1, and Fluo-4 fluorescence was acquired for 90 s. As a positive control, ionomycin (Sigma-Aldrich) was added to a final concentration of 16.4 mmol ml1 for the last 30 s of the measurement. Data were analysed using FlowJo software (BD Bioscience).

Intracellular serotonin staining

Stomach intraepithelial leucocytes were prepared as described above. Cells were incubated with ZombieFITC (1:500, BioLegend) for dead/live discrimination and 10 μg ml1 anti-CD16/32 antibodies (39, BioLegend) to block Fcγ receptors for 15 min at room temperature. After washing with PBS supplemented with 5% FCS (Sigma-Aldrich), cells were stained with CD45 BV785, CD117 APC and IgE BV421 (R35-72, BD Bioscience) for 20 min on ice and protected from light. After washing and centrifugation, cells were fixed and permeabilized using a FoxP3-intracellular staining kit (BioLegend) according to the manufacturer’s instructions. Cells were stained with 0.11 μg ml1 anti-5-HT (5HT-H209, Dako) or isotype control antibodies for 30 min, washed in PBS and stained with anti-mouse-IgG1 (RMG1-1, BioLegend) antibodies for 30 min before analysis with a BD LSRFortessa (Becton Dickinson).

Serological analysis

OVA-specific IgE and IgG1 were measured by enzyme-linked immunosorbent assay as previously described64. Anti-OVA IgG1 (L71, Biozol) and anti-OVA IgE (2C6, Invitrogen) were used as standards. Rat anti-mouse IgG1-HRP (1:2000, X56, BD Pharmingen) and rat anti-mouse IgE-HRP (1:2000, 23G3, SouthernBiotech) were used for detection. Serum samples were diluted 1:40000 (IgG1) and 1:10 (IgE). IL-4 and IL-6 were measured by LEGENDplex Mouse Th Cytokine (BioLegend) assay according to the manufacturer’s instructions.

Ca2+ imaging of full-thickness small intestine preparations

For ex vivo Ca2+ imaging, the ileum of immunized adult Wnt1|GCaMP3 mice or immunized and AAV9-transduced (pENN.AAV.CamKII.GCaMP6f.WPRE.SV40, Addgene) Cpa3+/+ and Cpa3Cre/+ mice was isolated. Tissues were opened along the mesenteric border and pinned flat in a Sylgard-lined dish containing Krebs solution (120.9 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl2, 1.2 mM NaH2PO4, 14.4 mM NaHCO3, 11.5 mM glucose and 2.5 mM CaCl2), bubbled with 95% O2/5% CO2 at room temperature. The luminal contents were cleared away with Krebs washes. Tissues were mounted over an inox ring and stabilized using a matched rubber O-ring65. Ring preparations were placed on a glass-bottomed dish and imaged using a ×20 objective on an inverted Zeiss Axiovert 200M microscope equipped with a monochromator (Poly V) and a cooled CCD camera (Imago QE) (TILL Photonics). Preparations were constantly superfused with carbogenated Krebs solution at room temperature using a local gravity-fed (±1 ml min−1) perfusion pipette. Krebs only, BSA (1% in Krebs) and OVA (1% in Krebs) were sequentially applied for 5 min each on to the mucosal surface using a perfusion pipette positioned above the imaged myenteric or submucosal plexus. Custom-written routines in Igor Pro (Wavemetrics)66 were used for analysis. Heatmaps display normalized fluorescence (Fi/F0) traces, and each row shows the fluorescence signal of an individual neuron under the control Krebs condition, followed by BSA (1%) and OVA (1%) mucosal perfusion. The signal of each neuron was normalized to baseline fluorescence under the Krebs condition. Traces depicted in the heatmaps are sorted top-down by the maximum amplitude of the signals detected. For percentages of activated neurons, regions of interest were drawn over each GCaMP-expressing neuron to calculate the average Ca2+ signal intensity normalized to the baseline (displayed as Fi/F0). Background subtraction was performed on some recordings where changes in background intensity were apparent. Neurons were considered to be active if at least one neuronal Ca2+ peak was detected during each 5 min recording period.


Vagotomy was performed as previously described67. In brief, both vagal trunks were transected below the diaphragm. To ensure transection of all small vagal branches, neural and connective tissue surrounding the oesophagus was removed. Pyloroplasty was performed to avoid gastric dilatation due to vagotomy. Control mice underwent a sham operation, in which vagal trunks were exposed but not cut and pyloroplasty was performed.

RNA isolation from stomach and small intestine

Naive, immunized and challenged (days 5, 7 or 11 of the two-bottle test; or 4×, 6× or 8× OVA gavage) BALB/c Cpa3+/+ and Cpa3Cre/+ mice were euthanized by CO2 asphyxiation. Stomach and small intestine pieces (4 cm proximal to the duodenum) were cut open, and food remnants, fat and Peyer’s plaques were removed. Tissues were immediately frozen in liquid nitrogen and ground in a mortar with a pestle. RNA isolation was performed according to the instructions of the PureLink RNA Mini kit (Invitrogen). Total RNA quality was determined by the RNA integrity number provided by the BioAnalyzer system (RNA 6000 Pico Kit, Agilent). Isolated RNA was stored at −80 °C until use.

RNA sequencing

Library preparation was performed with a TruSeq Stranded RNA Kit (Illumina) according to the manufacturer’s instructions. After library preparation, indexed samples were pooled and diluted to 2 nM with 2% PhiX spike in. The multiplexed library was then paired-end sequenced using NextSeq 1000/2000 P2 Reagents (200 Cycles) on the NextSeq 1000/2000 platform (conditions: Read1/Read2: 111 Cycles; Index1: 8 Cycles; Index2: 8 Cycles). Data were mapped using STAR aligner (v.2.5.2b)68, and reads were annotated using the FeatureCounts algorithm from the subread package (v.1.5.1)69. Both mapping and annotation were performed on Genome Reference Consortium Mouse Build 38 (GRCm38)70. Count data normalization and differential expression analysis were performed using DESeq2 (ref. 71), comparing immunized unchallenged mice with immunized mice of the same genotype challenged by either the two-bottle test or OVA gavage.

Principal component analysis was performed on read counts normalized by the variant stabilizing transformation included in the DESeq2 package, based on the top 500 most variable genes. For subsequent data analysis and visualization, shrinkage towards zero of log2 fold changes (lfcshrink) was computed using the apeglm implementation72. P values were adjusted using the Benjamini–Hochberg algorithm, and results were accepted as significant if adjusted P < 0.05.

Gene set enrichment analysis (GSEA) for each experimental group was performed on the complete dataset ranked with lfcshrink using ClusterProfiler73. All gene ontology (GO) terms related to ‘biological processes’ in the database74 were considered. Significantly (Benjamini–Hochberg adjusted P < 0.05) enriched GO terms were accepted for further processing. Based on the individual descriptions, enriched GO terms were manually annotated into four immune-related subgroups: innate immunity, adaptive immunity, mucosal immunity and chemotaxis (annotation of individual GO terms can be found in Supplementary Table 1). From subgrouped GO terms, core enrichment genes (genes that contribute most to the enrichment results for a GO term; GSEA documentation on the Broad website) were extracted and filtered (log2 fold change <−1 or log2 fold change >1.5 in at least one comparison). Fold changes of these genes were compared between experimental groups (Fig. 3a,c and Extended Data Fig. 7b,c).

Genes contained in the ‘hallmark inflammatory response’ gene set were retrieved from the Molecular Signatures Database (hallmark gene set collection: M5932, MSigDB)14, and their fold changes in wild-type mice under avoidance and non-avoidance conditions were compared.

In the volcano plots, horizontal lines indicate the significance threshold and vertical lines indicate the log2 fold change thresholds of −3 and 3, respectively. GSEA core enrichment genes are indicated in red, and manually curated mast-cell-related genes are depicted in blue (Fig. 3c–f). DEG with log2 fold change >3 in the stomach (Fig. 3c,d) and intestine (Fig. 3e,f) were filtered and displayed as a heatmap (Fig. 3g). In this heatmap, genes not significantly differentially expressed in other comparisons are depicted as white squares. Genes with significant (P < 0.05) changes in expression have coloured squares. The colour scale indicates log2 fold change.

Analysis of anxiety-like behaviour in Cpa3 Cre/+ mice

Anxiety-like behaviour was tested using an elevated plus maze, open field test and light–dark test. Mice are averse to brightly lit open areas. However, they have a natural drive to explore a perceived threatening stimulus. Low levels of anxiety lead to increased exploratory behaviour, whereas high levels of anxiety lead to less locomotion and more time spent in enclosed areas. Tests for anxiety-like behaviour were performed between 09:00 and 13:00. C57BL/6 Cpa3+/+ and Cpa3Cre/+ mice were brought into the behavioural room 30 min before behavioural testing began. All experimental setups were cleaned with soap and 70% ethanol at the end of the measurements.

The elevated plus maze consisted of an opaque-grey plastic apparatus with four arms (6 cm wide and 35 cm long), two open (illuminated with 100 lux) and two closed (20 lux), set in a cross from a neutral central intersection (6 × 6 cm) and elevated 70 cm above the floor. The mice were placed in the centre of the maze, and 5-min test sessions were digitally recorded and analysed with Sygnis Tracker software (Sygnis).

For the open field test, mice were placed in the centre of a bright open arena (40 cm wide, 40 cm long and 40 cm high; 290 lux), and their behaviour was monitored with a digital camera and ANY-maze video tracking system (Stoelting Co.) for 10 min.

The behavioural test box used for the light–dark test consisted of two compartments, a 29 × 21-cm (21 cm high; 300 lux illumination) lit compartment and a 15 × 21-cm (21 cm high; 10 lux illumination) dark compartment, connected by an opening at floor level allowing transition between the compartments. Mice were placed in the dark compartment, and their behaviour was monitored with a digital camera and Sygnis Tracker software for 10 min.

Analysis of general behaviour of Cpa3 Cre/+ mice

The LABORAS home cage observation system (Metris B.V.) consists of an adapted home cage placed on a carbon fibre platform that automatically detects behaviour-specific vibration patterns produced by the animal75. The LABORAS software (v.2.6.) processes the vibrations into various validated behaviours (climbing, grooming, locomotion immobility) and tracking information (distance travelled and speed). These behavioural parameters are automatically calculated as time duration or frequency counts. C57BL/6 Cpa3+/+ and Cpa3Cre/+ mice were individually placed in these calibrated cages under standard housing conditions with free access to food and water for a 24-h period. Mice were not habituated to the LABORAS cages before the experiments were started.


The following antibodies were used in flow cytometry: B220 FITC 1:50 (BD Pharmingen, RA3-6B2), CD3 BV421 1:200 (17A2, BioLegend), CD3 FITC 1:50 (17A2, BD Pharmingen), CD3 PE-Cy-7 1:25 (145-2C11, BD Pharmingen), CD11b PerCP-Cy5.5 1:400 (M1/70, eBioscience), CD11b BV421 1:400 (M1/70, BioLegend), CD11b PE-Cy-7 1:400 (M1/70, eBioscience), CD11c BV421 1:100 (N418, BioLegend), CD16/32 unconjugated 10 μg ml−1 (93, BioLegend), CD19 BV421 (6D5, BioLegend), CD19 APC 1:400 (1D3, BD Pharmingen), CD45 BV421 1:400 (30-F11, BioLegend), CD45 BV785 1:400 (30-F11, BioLegend), CD49b APC 1:100 (DX5, BD Pharmingen), CD90.2 APC-Cy7 1:400 (30-H12, BioLegend), CD117 PE 1:800 (2B8, eBioscience), CD117 APC 1:800 (2B8, BD Pharmingen), CD117 BV711 1:800 (2B8, BioLegend), FcεRI APC 1:200 (MAR-1, eBioscience), Gr-1 BV421 1:800 (RB6-8C5, BioLegend), Gr-1 BV605 1:200 (RB6-8C5, BioLegend), IgE PE 1:100 (RME1, BioLegend), IgE BV786 1:100 (RME-1, BD Pharmingen), IgE BV421 1:100 (RME-1, BD Pharmingen), Ly6G PerCP-Cy5.5 1:100 (1A8, BD Pharmingen), MHCII A700 1:100 (M5/114.15.2, eBioscience), Siglec-F BV421 1:100 (E50-2440, BD Pharmingen), Siglec-F PE 1:100 (E50-2440, BD Pharmingen), Ter119 BV421 1:200 (Ter119, BioLegend), 5-HT unconjugated 0.11 μg ml−1 (5HT-H209, Dako) and mouse-IgG1 PE 1:100 (RMG1-1, BioLegend).


Schematics shown in Figs. 4a and 5 and Extended Data Fig. 1a,e,j were made in Adobe Illustrator (v.25.0.1) using BioRender with permission to publish. The photograph of the IntelliCage was supplied by TSE Systems with permission to publish (Fig. 1b).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.