Abstract
Up to 20% of people worldwide develop gastrointestinal symptoms following a meal1, leading to decreased quality of life, substantial morbidity and high medical costs. Although the interest of both the scientific and lay communities in this issue has increased markedly in recent years, with the worldwide introduction of gluten-free and other diets, the underlying mechanisms of food-induced abdominal complaints remain largely unknown. Here we show that a bacterial infection and bacterial toxins can trigger an immune response that leads to the production of dietary-antigen-specific IgE antibodies in mice, which are limited to the intestine. Following subsequent oral ingestion of the respective dietary antigen, an IgE- and mast-cell-dependent mechanism induced increased visceral pain. This aberrant pain signalling resulted from histamine receptor H1-mediated sensitization of visceral afferents. Moreover, injection of food antigens (gluten, wheat, soy and milk) into the rectosigmoid mucosa of patients with irritable bowel syndrome induced local oedema and mast cell activation. Our results identify and characterize a peripheral mechanism that underlies food-induced abdominal pain, thereby creating new possibilities for the treatment of irritable bowel syndrome and related abdominal pain disorders.
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Data availability
The data for 16S rRNA gene sequences are available from the European Nucleotide Archive (https://www.ebi.ac.uk/ena/) under accession number PRJEB41204. All other relevant data generated and analysed during the current study are available upon reasonable request to the corresponding author. Source data are provided with this paper.
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Acknowledgements
We thank I. Croux and R. De Keyser for technical assistance; L. Beullens, A. W. Essilfie, H. Willekens and H. Volkaerts for nursing assistance in the studies involving human subjects; M. Rasulova for assistance with ELISpot plate read-out; and the Cell and Tissue Imaging Cluster for the use of the NIKON inverted TillVision microscope, epifluorescence microscope BX 41 Olympus and Zeiss LSM880—Airyscan—and Zeiss LSM 780—SP Mai Tai HP DS—confocal microscopes (Cell and Tissue Imaging Cluster, CIC, supported by Hercules AKUL/15/37_GOH1816N and FWO G.0929.15 to P. Vanden Berghe). Flow cytometry was done at the FACS Core facility (KU Leuven) and we thank P. Andrée Penttila and P. Kumar for technical assistance. We thank the National Reference Centre (NRC) for S. aureus of Université Libre de Bruxelles (ULB) for SAgs-positive S. aureus strains; and Genentech for the IgG2a anti-CD20 antibody and its isotype control. M.V.F., M.F.V. and D.B. were supported by FWO PhD fellowships (1110019N, 11C2219N and 1127415N, respectively). C. Breynaert is supported by the Clinical Research Fund, University Hospitals, Leuven. J.A.-L., M.M.W., P.J., N.S. and Y.A.A. were supported by FWO postdoctoral fellowships (12X9820N, 1248513N, 12R5219N, 12V3619N and 12H8220N, respectively). During this project J.A.-L. was also previously supported by an FWO PhD fellowship (11Y2116N). T.B.F. and H.-R.R. were supported by SFB-TRR156-A07. S.D.B. is supported by NIH grant NIAID R01 AI125567 and an endowment from the Crown Family Foundation. G.E.B. was funded by a KU Leuven university grant (Global Opportunities for Associations GOA 14.011, C14/18/086) and an FWO grant (G0A9516N).
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Authors and Affiliations
Contributions
J.A.-L., M.V.F., A.D.-S., M.M.W. and G.E.B. planned and designed the experiments. J.A.-L. and M.V.F. performed mouse infections and pharmacological treatments and carried out in vivo animal protocols. J.A.-L., M.V.F., I.A. and M.C.-E. performed immunoglobulin ELISAs. J.A.-L., M.C.-E. and S.U.M. performed diarrhoea-development experiments. J.A.-L. recorded VMRs and compliance, except for pyrilamine treatment and in Hrh1−/− mice, which were performed by M.V.F. E.P. performed afferent nerve recordings. J.A.-L. performed colonic permeability assessments, assisted by R.F. J. Si and J.R. performed analysis of murine microbiota. M.V.F. and S.T. performed histopathology assessments in mouse samples. N.S. performed cytokine quantification by cytometric bead array. J.A.-L., I.A. and N.F. performed RNA extraction and quantitative PCR experiments. M.F.V., M.V.F., I.A., N.F. and G.B. performed FACS experiments, assisted by G.M., S.I.M., C. Bosteels and B.N.L. ELISPOT assay was performed by L.D. and A.D.-S. C.L.-L. and J.J.-P. performed patch-clamp experiments, assisted by D.E.R. J.A.-L. performed Ca2+ experiments, assisted by Y.A.A. and K.T. J.A.-L. performed bacterial DNA extraction from faecal samples. P.J. and M.F.V. performed immunofluorescence staining of human samples and total corrected cell fluorescence analysis. P.J. and M.C.-E. performed and analysed immunofluorescence staining of mouse samples and mast cell live imaging analysis. R.B., G.E.B., K.V.B. and D.B. performed food antigen injections and collection of biopsies. J.A.-L. and D.B. performed the analysis of the reactions to food antigens. C. Breynaert and R.S. performed the allergy work-up in humans. A.D.-S. designed, performed and analysed protease activity assays. A.D.-S. and M.N. evaluated histamine presence in mouse and human samples, assisted by D.C. and P.A. IgH sequence analysis of IgE+ clones was performed by R.A.H., assisted by S.D.B. T.B.F. and H.-R.R. provided Cpa3Cre/+ mice, and J. Strid provided Igh7−/− mice. S.H. and F.A.R. provided experimental support. J.A.-L., M.V.F., M.F.V., A.D.-S., D.E.R., S.J.V., M.M.W. and G.E.B. reviewed data. J.A.-L., A.D.-S. and G.E.B. wrote and revised the manuscript. All other authors corrected and approved the final version of the manuscript. M.F.V. and P.J. contributed equally to this manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Extended analysis of the OVA-specific immune response and VHS in post-infectious mice.
a, b, diarrhoea development quantification by (a) water content in faeces and (b) whole-gut transit time upon gavage of carmine red dye in OVA/sham + OVA, OVA/infected + OVA (n = 10/group) mice. c, quantification of OVA-specific IgE in intestinal homogenates of OVA/sham + OVA, saline/infected + OVA, OVA/infected + saline and OVA/infected + OVA mice (n = 9, 10, 10 and 9, respectively) at 7 weeks post-infection. d, ear-swelling after intradermal injection of OVA in OVA/sham + OVA (n = 6), OVA/infected + OVA (n = 10), at 7 weeks post-infection, and OVA-allergy mice (n = 6, 10 and 6, respectively). e, f, VMR to colorectal distention in (e) OVA/sham + OVA, OVA/infected + saline, saline/infected + OVA and OVA/infected + OVA mice (n = 12, 11, 11 and 13, respectively) and (f) tracing of a electromyographic response to 20-μL-, 40-μL-, 60-μL- and 80-μL-volume colorectal distention in a OVA/infected + OVA mouse at baseline and 7 weeks post-infection. g, VMR to colorectal distention in OVA/infected + OVA (n = 7) and OVA/infected = Saline (n = 7 and 5, respectively) mice at baseline (BL), 7 weeks post-infection (PI) and after 1, 2, 3 and 4 weeks after stopping oral OVA or saline re-exposure, respectively. h, colonic permeability in BALB/c mice expressed as passage of fluorescein sodium (left) and transepithelial resistance (right) in OVA/sham + OVA, saline/infected + OVA, OVA/infected + saline and OVA/infected + OVA mice (n = 8, 11, 12 and 8, respectively) at 7 weeks post-infection. i, scheme illustrating the post-infectious protocol with prior OVA tolerization. j, VMR to colorectal distention in mice OVA-tolerized (high-dose) + saline/infected + OVA, OVA-tolerized (high-dose) + OVA/infected + OVA, OVA-tolerized (low-dose) + saline/infected + OVA and OVA-tolerized (low-dose) + OVA/infected + OVA (n = 6, 9, 6 and 8, respectively). k, l, VMR to colorectal distention in mice OVA/infected repeatedly gavaged with BSA compared to OVA (n = 6 and 10, respectively). m, scheme illustrating the post-infectious protocol in mice treated with anti-IgE antibody. n, VMR to colorectal distention in OVA/infected + OVA mice treated with anti-IgE antibody or control antibody (n = 8/group). o, colonic permeability expressed as passage of fluorescein sodium (left) and transepithelial resistance (right) of (l) VHS mice treated with anti-IgE antibody or control antibody (n = 8/group). p, VMR to colorectal distention in OVA/infected + OVA mice with WT or Igh7−/− background (n = 10/group). q, colonic permeability expressed as passage of fluorescein sodium (left) and transepithelial resistance (right) of OVA/infected + OVA mice with Igh7−/− background or WT mice (n = 10/group) at 7 weeks post-infection. r, scheme illustrating the protocol in mice that received monoclonal OVA-specific IgE antibody. s, VMR to colorectal distention in naïve mice treated with monoclonal OVA-specific IgE antibody or monoclonal Dinitrophenyl (DNP) antibody (n = 7 and 6, respectively). Two-tailed Mann–Whitney test in a and b for every time point, and in o and q (left); two-tailed t-test for q (right). Kruskal–Wallis test (Dunn’s multiple-comparisons test) in c and d. One-way ANOVA (Sidak’s multiple-comparisons test) in h. Two-way repeated ANOVA (Sidak’s multiple-comparisons test) in e, g, j–l, n, p and s. Data shown as Box-and-whiskers (centre line, median; box, 25th-75th percentiles; whiskers, 10th-90th percentiles) in a, b and d and median ± IQR in c, e, g, h, j–l, n-q and s. VMR, visceromotor response; BL, baseline; w, week; d; day.
Extended Data Fig. 2 Changes in microbiota composition are not likely to be associated with the development in VHS.
a, changes in the gut microbiome composition over time studied by ordination (PCA) plot before the infection, 10 days after infection (R2 = 0.11, p-value = 0.02), and after OVA gavage (R2 = 0.18, p-value = 0.002) between OVA/infected and saline/infected (n = 8/group) mice. The numbers in brackets indicate the variation explained by each axis (PCA: principal component analysis). b–d, drivers of variation in the murine intestinal community after OVA re-exposure. b, Comparison of mean abundance of taxa between OVA- and saline-infected mice. Bacterial abundances were computed using centred log-ratio transformation (CLR; two-sided Mann–Whitney test, FDR <0.2). c, ordination biplot of PCA analysis showing the significant contributors in the community variation. Arrows indicate the relative importance of each genus (permutation-based P < 0.05). d, abundance of genus Robinsoniella, Eisenbergiella, Senegalimassilia, Murimonas, Clostridium_XVIII and Stomatobaculum by group and VHS (two-sided Mann–Whitney test, FDR >0.1). e, scheme illustrating the post-infectious protocol in mice treated with antibiotics. f, g, VMR to colorectal distention in VHS (OVA/infected + OVA) mice treated with antibiotics or control, and compared to OVA/infected + saline mice (n = 10, 7 and 6, respectively) (g depicts the VMM response at 7 weeks post-infection). In d, VHS threshold was established based on the 95th percentile of visceromotor responses at baseline (AUC >4.8). There was no significant association between VHS and bacterial abundance in any group (two-tailed Mann–Whitney test). X-axis indicates absence (0) and presence (1) of VHS. Two-way ANOVA 9(Sidak’s multiple-comparisons test) in f and g. Data shown as box-and-whiskers ± 1.5 IQR in d and as median ± IQR in f and g. VMR, visceromotor response; BL, baseline; w, week.
Extended Data Fig. 3 Analysis of ‘low grade’ inflammation in colonic samples of post-infectious mice.
a, Representative H&E-stained colonic sections from OVA/sham + OVA, OVA/infected + saline, saline/infected + OVA and OVA/infected + OVA at 7 weeks post-infection. b, thickness of the colonic muscularis (left), length of the crypts (middle) and distance between crypts (right), measured in OVA/sham + OVA, saline/infected + OVA (n = 6), OVA/infected + saline and OVA/infected + OVA mice (n = 8, 6, 8 and 8, respectively) at 7 weeks post-infection. c–k, concentration of (c) IFN-γ, (d) TNF, (e) IL-6, (f) KC (murine IL-8 homologue), (g) MCP-1, (h) IL-17A, (i) IL-13, (j) IL-4 and (k) IL-10 assessed in colonic supernatant and (l) IL-13 and (m) IL-5 in colon-draining lymph nodes of OVA/sham + OVA, saline/infected + OVA, OVA/infected + saline and OVA/infected + OVA mice (n = 8, 11, 12 and 8, respectively, for c–k; n = 7, 7, 9 and 9, respectively for l and m). n–q, immune cell quantification along the intestinal tract: (n and p) gating strategy; (o) quantification of population of CD11b+Ly6G+SiglecF- neutrophils, CD11b+SiglecF+ eosinophils, CD117+FcRεI+ mast cells, CD3+ T cells, CD3+CD4+ T cells, CD3+CD4+Foxp3+ T cells and (q) Helios-Nrp1loFOXP3+ pTregs in the colon and small intestine in OVA/sham + OVA, saline/infected + OVA, OVA/infected + saline and OVA/infected + OVA mice (n = 7, 6, 7 and 7, respectively, for o (colon); 9, 8, 9 and 10, respectively, for o (small intestine); 9, 9, 8 and 10, respectively, for q (Helios-Nrp1loFOXP3+ pTregs in colon); 9, 9, 8 and 10, respectively, for q (Helios-Nrp1loFOXP3+ pTregs in small intestine);. One-way ANOVA (Sidak’s multiple-comparisons test) in b–m, o and q. Data shown median ± IQR in b–m, o and q.
Extended Data Fig. 4 Extended analysis of mast cell activation as key players in the development of OVA-induced VHS in post-infectious mice.
a–c, avidin-fluorescence intensity over time (expressed as TCCF) in MC from OVA/sham + OVA, saline/infected + OVA OVA/infected + saline and OVA/infected + OVA mice (n = 222 [7 mice], 145 [6 mice], 202 [8 mice] and 239 [9 mice], respectively), (a) shown as percentage at times = 0, 7, 14, 21 and 28 min, (b) micrographs and (c) shown at time = 28 min as median per mouse. In b, arrows point to representative MC showing degranulation (yellow, 28 min) compared to baseline (white, 0 min). d, representative image of double-staining using DAPI and avidin in fixed colonic tissue from healthy mice (n = 3). e, Histamine quantification in supernatant collected from OVA/sham + OVA, saline/infected + OVA, OVA/infected + saline and OVA/infected + OVA (n = 10/group) at 7 weeks post-infection. f, VMR to colorectal distention in OVA/infected + OVA mice (d) treated with doxantrazole or vehicle (n = 14 and 11, respectively). g, colonic permeability expressed as passage of fluorescein sodium (left) and transepithelial electrical resistance (right) of OVA/infected + OVA mice treated with doxantrazole or vehicle (n = 8/group) at 8 weeks post-infection. h, quantification of population of CD117+FcRεI+ mast cells in intestinal lamina propria from Cpa3Cre/+ and WT mice (n = 3/group). i, VMR to colorectal distention in OVA/infected + OVA mice with Cpa3Cre/+ background or WT littermates (n = 13 and 12, respectively). j, colonic permeability expressed as passage of fluorescein sodium (left) and transepithelial electrical resistance (right) in OVA/infected + OVA mice with Cpa3Cre/+ background or WT littermates (n = 12/group) at 9 weeks post-infection. k, scheme illustrating the post-infectious protocol in mice treated with anti-CD20 antibody and bortezomib. l, anti-CD20 (5D2 clone) antibody and bortezomib depleted CD19+ Ly6K+ plasma cells (left) and B220+ IgM+ B cells (right) from the colon of mice compared to control antibody/vehicle and naïve mice (n = 2, 1 and 4, respectively) and (m) gating strategy used to validate B cell and plasma cell depletion. n, quantification of OVA-specific IgE in colon samples homogenates from OVA/infected + OVA mice treated with anti-CD20 antibody and bortezomib or control antibody and vehicle (n = 12 and 9, respectively). o, VMR to colorectal distention in OVA/infected + OVA mice treated with anti-CD20 antibody/bortezomib or control antibody/vehicle (n = 3 and 4, respectively). Mixed-effects model (Dunnett’s multiple-comparisons test) in a and e. One-way ANOVA (Sidak’s multiple-comparisons test) in c. two-way repeated measures ANOVA (Sidak’s multiple-comparisons test) in f, (I) and o. Two-tailed Mann–Whitney in g, h, j and n. Data are shown as median ± IQR in a, e–j, l, n and o, and box-and-whiskers (centre line, median; box, 25th and 75th percentiles; whiskers, 10th and 90th percentiles) in c. VMR, visceromotor response; BL, baseline; w, week.
Extended Data Fig. 5 H1R mediates OVA-induced VHS in post-infectious mice.
a, representative tracing and Fura-2 ratiometric fields of DRG neurons (TRPV1+ neuron [right] indicated by arrowhead). b, c, VMR to colorectal distention in OVA/infected + OVA mice (a) treated with vehicle and pyrilamine (n = 9 and 8, respectively) and (b) with WT and Hrh1−/− background (n = 13 and 9, respectively). Two-way repeated measures ANOVA (Sidak’s multiple-comparisons test) in b and c. Data are shown as median ± IQR.
Extended Data Fig. 6 SEB induces MC-mediated OVA-induced VHS upon comparable to C. rodentium infection.
a, scheme illustrating the SEB protocol. b, VMR to colorectal distention in saline/SEB + OVA, OVA/SEB + OVA and OVA (low dose) + OVA/SEB + OVA mice (n = 10, 14 and 4, respectively). c, colonic permeability expressed as passage of fluorescein sodium (left) and transepithelial electrical resistance (right) of saline/SEB + OVA and OVA/SEB + OVA (n = 8 and 7, respectively) mice. d, heat-map of the gene expression of inflammatory genes and mast-cell-related genes in OVA/saline + OVA (a), saline/SEB + OVA (b) and OVA/SEB + OVA mice (c) (n = 21, 7 and 7, respectively; except for Tryptase α/β-1: n = 7/group). e, VMR to colorectal distention in SEB-VHS mice treated with doxantrazole or vehicle (n = 8 and 9, respectively). f, colonic permeability expressed as passage of fluorescein sodium (left) and transepithelial electrical resistance (right) of SEB-VHS mice treated with doxantrazole or vehicle (n = 8 and 7, respectively). g, VMR to colorectal distention in OVA/SEB + OVA mice with Cpa3Cre/+ background or WT littermates (n = 5 and 6, respectively). h, colonic permeability expressed as passage of fluorescein sodium (left) and transepithelial electrical resistance (right) of OVA/SEB + OVA mice with Cpa3Cre/+ background or WT littermates (n = 5 and 6, respectively). i, quantification of OVA-specific IgE in colon homogenates of saline/SEB + OVA and OVA/SEB + OVA mice (n = 8 and 7, respectively). j, ear-swelling after intradermal injection of OVA in saline/SEB + OVA, OVA/SEB + OVA and OVA-allergy mice (n = 9, 7 and 6, respectively) mice. k, % of HV and IBS patients (n = 64 and 84, respectively) positive for S. aureus (left) and SAg-encoding S. aureus (right) in faecal samples. Numbers below the bars represent the ratio of positive and total HV and patients. Two-way repeated ANOVA (Sidak’s multiple-comparisons test) in b, e and g. Two-tailed unpaired t-test in c and f. Two-tailed Mann–Whitney test in h and i. Kruskal–Wallis test (Dunn’s multiple-comparisons test) in d and j. Two-sided Fisher’s exact test in k. Data shown as median ± IQR in b, e–i and box-and-whiskers (centre line, median; box, 25th-75th percentiles; whiskers, 10th-90th percentiles) in j. VMR, visceromotor response; BL, baseline; w, week. In d, Tpsab1, Il4, Il6 and Il10 gene expression differences were statistically significant in OVA/SEB + OVA vs. OVA/saline + OVA (adjusted P = 0.0005, 0.0004, 0.0163 and 0.0039, respectively).
Extended Data Fig. 7 Extended analysis of mast cells and IgE in human samples.
a, measurement of trypsin-like activity in supernatants of rectal biopsies from HV and IBS patients (n = 13 and 48, respectively) in basal conditions. b, c, number of (a) MC (CD117+) and (b) IgE+-MC (CD117+ IgE+) quantified in mucosal rectal biopsies from HV and IBS patients (n = 15 and 22, respectively). d, quantification of CD117+ IgE+ cells (MC) at ≤ 5 μm from nerve fibres (β tubulin III+ cells) in HV and IBS patients (n = 15 and 17, respectively). e, correlation between the IgE TCCF and max mucosal diameter after food-antigen injection in IBS patients (n = 7). f, number of IgE+ clones was quantified in mucosal rectal biopsies cDNA from HV and IBS patients (n = 10 and 20, respectively) using deep sequencing. Two-tailed Mann–Whitney test in a–d and f. two-tailed Pearson’s correlation in e. Data are shown as median ± IQR in a–d, and as box-and-whiskers (centre line, median; box, 25th and 75th percentiles; whiskers, 10th and 90th percentiles) in f. HPF = high power field.
Extended Data Fig. 8 Graphical representation of the mechanism proposed: local immune response to dietary antigens triggered by bacterial infection leads to food-induced abdominal pain.
Bacterial infection (or bacterial toxins, SEB) can trigger break of oral tolerance to food antigens leading to food-induced VHS upon food-antigen re-exposure. OVA-specific IgE antibodies bind to and sensitizes tissue-resident mast cells, which are activated upon re-exposure to OVA during feeding and release mediators that sensitize afferent neurons via H1R-mediated pathway. Components of this figure were created using Servier Medical Art templates, which are licensed under a Creative Commons Attribution 3.0 Unported License; https://smart.servier.com.
Supplementary information
Supplementary Table 1
A list of primer sequences for gene detection by RT-qPCR.
Video 1
Injection of food antigens in the rectosigmoid mucosa. Representative injection of food-antigen solutions in the rectosigmoid mucosa of participants.
Video 2
Mucosal edema reaction to the positive-control histamine. Representative reaction 12-min after histamine injection.
Video 3
Mucosal edema reaction to food antigens in HV Representative reaction 12-min after food-antigen injection (milk) in HV.
Video 4
Mucosal edema reaction to food antigens in IBS patients. Representative reaction after 12-min food-antigen injection (milk) in IBS patients.
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Aguilera-Lizarraga, J., Florens, M.V., Viola, M.F. et al. Local immune response to food antigens drives meal-induced abdominal pain. Nature 590, 151–156 (2021). https://doi.org/10.1038/s41586-020-03118-2
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DOI: https://doi.org/10.1038/s41586-020-03118-2
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